Method for making glucagon and insulin restore normal balance

ABSTRACT

The present invention relates to a method for restoring a balance between glucagon and insulin to normal, comprising administering an effective amount of plasminogen to a subject; furthermore, the present invention relates to a medicament for restoring a balance between glucagon and insulin to normal.

RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national stage filing ofInternational Application No. PCT/CN2017/089066, filed on Jun. 19, 2017,which claims priority from International Application No.PCT/CN2016/110171, filed on Dec. 15, 2016. The contents of theseapplications are incorporated herein by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 14, 2019, isnamed BCLS_006US_SEQ.txt and is 47245 bytes in size.

TECHNICAL FIELD

The present invention relates to a method for restoring a balancebetween glucagon and insulin to normal, comprising administering aneffective amount of plasminogen to a subject; furthermore, the presentinvention relates to a medicament for restoring a balance betweenglucagon and insulin to normal.

BACKGROUND ART

Diabetes mellitus (DM) is a common genetically predisposed abnormalglucose metabolism disease with endocrine disorder, and is caused byabsolute or relative insufficient insulin secretion. In 2015, there were415 million patients with diabetes mellitus worldwide, and the number ofpatients with diabetes mellitus is expected to reach 642 million by2040^([1]). Diabetes mellitus is one of the major diseases thatseriously endanger human health.

The main manifestations of diabetes mellitus are abnormal glucosemetabolism and metabolic disorders of substances such as fats andproteins; furthermore, long-term hyperglycemia may lead to seriousdiabetic complications, including microvascular complications, diabeticnephropathy, diabetic cardiomyopathy, diabetic neuropathy, diabeticdermopathy, diabetes mellitus with infections, etc. Among them, diabeticnephropathy and diabetic neuropathy have a great impact on the qualityof the life of patients, and are severely harmful.

Clinically common diabetes mellitus can be divided into four types: type1 diabetes mellitus (T1DM), type 2 diabetes mellitus (T2DM), gestationaldiabetes mellitus, and special types of diabetes mellitus. Among them,patients with T1DM and T2DM are the most common, while patients withgestational diabetes mellitus and special types of diabetes mellitus arerelatively few.

T1DM is believed to be associated with genetic factors, environmentalfactors (such as viral infections, diabetogenic chemicals, and dietaryfactors) and autoimmune factors. Studies have shown that there are atleast 17 gene loci associated with T1DM, which are located on differentchromosomes. In terms of environmental factors, environmental factorsthat affect the onset of T1DM comprise viral infections, diabetogenicchemicals, and dietary factors, in which viral factors are the mostimportant. By far, mumps, rubella virus, cytomegalovirus and the likehave been found to be associated with pathogenesis of T1DM. Themechanism is that the viruses can directly destroy pancreatic islet βcells, and after the viruses damage the pancreatic islet β cells,autoimmune reactions are triggered, which cause further damage to thepancreatic islet β cells. Diabetogenic chemicals such as alloxan,streptozotocin (STZ) and pentamidine act on pancreatic islet β cells,leading to destruction of the pancreatic islet β cells. The autoimmunefactors comprise humoral immunity and cellular immunity. Humoralimmunity is manifested by the presence of multiple autoantibodiesagainst pancreatic islet β cells in the blood circulation of a patient.The main manifestation of cellular immunity is that abnormal expressionof HLA-DA antigen and overexpression of IL-2 receptor and pancreaticislet cell surface HLA class 1 antigens can be observed on surfaces ofpancreatic islet inflammatory infiltrating cells and pancreatic islet βcells, and the ratio of CD4+/CD8+ in the peripheral blood and the levelsof IL-1, TNF-α, and INF-γ are elevated. The pathological changes causedby these factors focus on the destruction of the pancreatic isletβ-cells, resulting in an absolute decrease in the level of insulin inthe body, thereby causing T1DM, and therefore T1DM is considered to bean autoimmune disease.

T2DM is a polygenic disease, and is generally considered to bemulti-sourced, wherein environmental factors and genetic factors worktogether to cause insulin resistance; and the manifestation of T2DM isthat insulin at a concentration the same as the normal level cannotfunction normally due to the resistance in the body. Accordingly, inorder to achieve the normal blood glucose level, the body willexcessively secrete insulin to alleviate the “low-efficiency” state ofinsulin in service, and if it continues this way, the requirements forthe pancreatic islet β cells are getting higher and higher, ultimatelycausing damage to the pancreatic islet β cells themselves due to“overwork”, thus developing into absolute insulin deficiency.

Pathogenesis of DM

The pathogenesis of DM is complex, and is mainly related to familygenetic predisposition, ethnic heterogeneity, insulin receptordeficiency, impaired insulin receptor substrate, up-regulation ofprotein tyrosine phosphatase-related genes, excessive immuneinflammatory response, lipotoxicity, oxidative stress, impairedmitochondria etc.^([2-3])

1. Free Fatty Acids

Elevated levels of free fatty acids are one of the causes of insulinresistance and also one of the important characteristics of insulinresistance. Under the influence of genetic factors or environmentalfactors, the level of free fatty acids in the blood increases, and whenit exceeds the storage capacity of adipose tissues, insulin resistanceoccurs. Studies have shown that long-term high-fat diets lead topancreatic islet β cell dysfunction, because high-fat diets not onlytrigger peripheral insulin resistance, but also increase the abdominalfat content and reduce the capacity of insulin to inhibit lipolysis,thereby promoting an increase in the content of free fatty acids, whichin turn inhibits the phosphorylation of tyrosine sites in the insulinreceptor and the insulin receptor substrates IRS-1 and IRS-2, therebyinhibiting the activity of P13K, which results in the insulin signaltransduction pathway being hindered, thereby forming insulin resistance.

2. Inflammatory Response

1) Inflammation and Insulin Resistance

T2DM is a mild, non-specific inflammatory disease. Studies of recentyears have shown that the main mechanism of inflammation leading toinsulin resistance is that there is a cross-effect between inflammatoryfactors and the signal transduction of insulin receptor substrates: onthe one hand, an inflammatory factor resulting from non-specificinflammation hinders the IRS/PI3K signaling pathway, and on the otherhand, a series of kinases activated by the inflammatory factor inducephosphorylation of serine and threonine sites in IRS, which hindersnormal tyrosine phosphorylation, ultimately resulting in the insulinsignal transduction capacity being decreased and insulin resistancebeing induced^([2-3]).

In a target cell, the binding of insulin to a receptor thereof canactivate the receptor, then the signal transduction pathway in the cellresults in a series of intracellular transduction molecules andenzymatic cascade reactions to complete the stepwise transmission andamplification of the signal in the cell, and finally, the signal ispassed to a target organ to produce a series of biological effects.There are two main signal transduction pathways, one beingIRS-1-PI3K-PKB/AKT pathway and the other being mitogen-activated proteinkinase (Shc/Raf/MAPK) pathway. In the first pathway, firstly insulinbinds to a receptor thereof under the stimulation of exogenous insulinand/or glucose, thereby activating an endogenous tyrosine kinase of thereceptor. The activated tyrosine kinase induces tyrosine sitephosphorylation in the insulin receptor substrate IRS while achievingphosphorylation of the tyrosine kinase itself. The activated IRSmigrates to the cell membrane, phosphotyrosine is anchored to the IRStyrosine kinase via a phosphotyrosine binding domain (PTB), and thetyrosine-phosphorylated IRS recruits regulatory subunit P85 of PI3K viaan SH2 domain. P85 binds to a phosphoinositol 3-phosphate molecule andconverts phosphatidylinositol monophosphate (PIP) tophosphatidylinositol diphosphate (PIP2) and phosphatidylinositoltriphosphate (PIP3), both of which are second messengers of insulin andother growth factors, and are anchor sites for downstream signalingmolecules phosphoinositide-dependent protein kinase-1 (PDK1) and/or somesubtypes of protein kinase c (PKC). PDK1 can activate protein kinase B(PKB, also known as Akt) and an atypical PKC subtype. The activated PKBon the one hand inactivates glycogen synthase kinase-3 (GSK3) by meansof serine/threonine phosphorylation and on the other hand activates themammalian target of rapamycin (mTOR) protein kinase, thereby inducingphosphorylation activation of 70ku-S6 kinase (p70S6K) downstream. ThemTOR protein kinase can act as an “ATP receptor” and activates p70s6Kwithout Ca₂ ⁺/cAMP, thereby achieving controlled protein synthesis,enhanced gene transcription, and facilitation of pancreatic islet β cellhypertrophy, as well as other biological effects. PKB can directlyinduce the phosphorylation of serine/threonine in certain transcriptionfactors to promote the occurrence of cell mitosis^([4-5]). In the secondpathway, the activation of Ras may be achieved via two pathways. 1)Activated insulin receptor activates IRS-2 protein, and the IRS-2protein can transmit the signal to the adaptor protein growth factorreceptor binding protein 2 (Grb2), which in turn interacts with asignaling protein GDP/GTP exchange factor (mSOS), and can in turnactivate inactivated Ras-GDP into Ras-GT to achieve the activation ofRas. The direct action of the insulin receptor phosphorylates tyrosinein signaling protein Shc, and then Shc binds to Grb2 to activate Ras viathe mSOS pathway. Activated Ras-GTP recruits Raf serine kinase, whichsequentially phosphorylates MAPK kinase and MAPK. The activated MAPK mayactivate other protein kinases to participate in processes such asinducing gene transcription, and regulating apoptosis^([6]).

By far, it has been confirmed that the serine residue of IRS-1 may bephosphorylated by various inflammatory kinases such as c-Jun N-terminalkinase (JNK), IκB kinase β (IκKβ) and protein kinase C (PKC)-θ. Radioimmunoassay shows that serine site 307 is the major site for thephosphorylation of IRS-1 by JNK, and its mutation causes JNK-inducedIRS-1 phosphorylation and the inhibitory effect of TNF oninsulin-induced IRS-1 tyrosine phosphorylation to disappear. JNK reducesthe phosphorylation of tyrosine in the insulin receptor substrate byphosphorylating serine 307 of IRS-1, thereby inhibiting insulin signaltransduction^([7]). Hiorsumi et al. found that the activity of JNK wassignificantly increased in the liver, muscle, and adipose tissues ofdiet-induced obese mice and ob/ob mice. Gene knockout (JNK1−/−) canattenuate insulin resistance in the diet-induced obese mice andalleviate obesity, hyperglycemia, and hyperinsulinemia in the ob/obmice. The level of phosphorylation of serine site 307 of IRS-1 in theliver tissue of the obese mice was higher than that of lean mice;however, no increase was found in the knockout (JNK1−/−) obese mice; itcan be seen that the serine site 307 of IRS-1 was the target at whichthe JNK acts in vivo^([8]). Studies have shown that in a model of TNFαstimulation-induced hepatocyte insulin resistance, JNK inhibitors cancompletely block the phosphorylation of serine 307. IκKβ can affectinsulin signal transduction via at least two pathways, i.e., by directlyinducing the phosphorylation of Ser307 of IRS-1 or by thephosphorylation of IκB, thereby activating NF-κB, which indirectlyinduces insulin resistance by stimulating the expression of variousinflammatory factors.

Inflammatory responses are defensive responses of the human immunesystem against infections, tissue damages and stress responses afterthese injuries occur, and are also involved in etiology or pathogenesisof diabetes mellitus, cardiovascular diseases and tumors.

As early as in 1993, Hotmamisligil et al.^([9]) demonstrated throughanimal experiments that insulin-resistant obese rats had high levels ofpro-inflammatory cytokines and TNF-α in adipose tissues. Since then,many researchers have begun to explore the relationship betweeninflammation and obesity and the relationship between inflammation andinsulin resistance, and explore the molecular pathogenesis. In 2006,Hotmamisligil^([10]) first proposed a new medical definition, i.e.metabolic inflammation, to emphasize that this low-grade, chronicsystemic inflammation is mainly caused by excess nutrients andmetabolites. Metabolic inflammation may have molecular and signaltransduction pathways similar to those for typical inflammations; unliketypical inflammations that we have known in the past, metabolicinflammation does not have the symptoms of redness, swelling, heat,pain, and dysfunction. Under normal circumstances, the internalenvironment of the body is in a steady state, and inflammations andmetabolisms maintain dynamic equilibrium states respectively ortherebetween. In case of metabolic disorders in an body, such anequilibrium in the body is broken, causing imbalance of the immunesystem, triggering an inflammatory signal transduction pathway, therebyprompting the body to release a series of inflammatory factors. Some ofthe inflammatory factors even amplify antoinflammatory responses to forman inflammatory waterfall effect, which further develops insulinresistance in the body, thus leading to the occurrence of metabolicsyndrome.

Studies have shown that TNF-α is closely related to metabolic syndrome.TNFs, also known as dyscrasia, are mainly produced by activatedmacrophages, natural killer (NK) cells and T lymphocytes, wherein theTNF secreted by macrophages is called TNF-α, and lymphotoxin secreted byT lymphocytes is called TNF-β. The biological activity of TNF-α accountsfor 70%-95% of the overall activity of TNFs, and therefore, usuallyreference to TNF at present is mostly reference to TNF-α. After years ofresearch and discussion, it has been confirmed that TNF-α is associatedwith various diseases such as insulin resistance, autoimmune diseases,tumors, and chronic hepatitis B. TNF-α plays a crucial role in onset anddevelopment of insulin resistance. Swaroop et al.^([11]) concluded bydetecting the level of serum TNF-α in 50 patients with T2DM that theTNF-α levels are elevated in the patients with T2DM and aresignificantly associated with BMI, fasting insulin level, andhomeostatic model assessment insulin resistance index (HOMA-IR),suggesting that TNF-α plays an important role in pathogenesis of T2DM.It has also been pointed out in additional studies that TNF-α caninhibit the phosphorylation of the insulin receptor, and when thephosphorylation of the insulin receptor is inhibited, the expression ofthe gene of glucose transporter can be reduced, thereby reducing theactivity of lipoprotein lipase, ultimately leading to lipolysis^([12]).

2) Inflammation and Pancreatic Islet β Cell Apoptosis

A chronic, low-grade inflammatory response is closely related topancreatic islet β cell dysfunction. Pancreatic islet β cell dysfunctioncaused by a decrease in the number of β cells is another important causeof the pathogenesis of T2DM, and β cell apoptosis is the most importantcause of the decrease in the number of the β cells. Due to genetic ordietary reasons, patients with T2DM are susceptible to insulinresistance; furthermore, in case of patients with elevated bloodglucose, hyperglycemia can promote production of IL-6 which can not onlyreduce expression of GLUT4, reduce transport of glucose by fat cells,hinder glycogen synthesis, and reduce insulin sensitivity, but can alsopromote secretion of IL-6 by pancreatic islet cells, causing a viciouscircle. Hyperglycemia induces the production of a large amount of IL-1β,which results in pancreatic islet cell apoptosis by activating pathwayssuch as NF-κB, MAPK, Fas and NO, and there are cross-facilitations ofvarious inflammatory pathways to aggravate the apoptosis of pancreaticislet cells, which eventually leads to pancreatic islet functionfailure^([13]). In addition, IL-1β can also mediate interactions ofleukocytes, and mutually interact and restrict with other cytokines suchas IFN-γ and TNF-α, and play an important role in the process of a βcell injury. Dyslipidemia in T2DM causes an increase in the level ofhormonal substances such as leptin and that of IL-6. Leptin can increasethe release of IL-1β to induce β cell apoptosis, and can also negativelyregulate insulin secretion^([14]). In addition to causing insulinresistance, ROS also has an effect on the injury of pancreatic islet βcells, and under oxidative stress, the expression of insulin genetranscription factors, and insulin binding sites are remarkably reduced,thereby affecting the production and secretion of insulin. Otheradipocytokines such as TNF-α and leptin may also reduce the function ofthe β cells^([15]). The combined action of these cytokines causes moreremarkable damage to the function of the pancreatic islet β cells. Inaddition, some inflammatory factors may also act on the key part ofinsulin receptor substrate 2 to phosphorylate serine/threonine, whichresults in accelerated degradation of insulin receptor substrate 2 andpromotes apoptosis of pancreatic islet β cells.

3. Oxidative Stress

Studies have shown that oxidative stress is an important factor in theonset and development of T2DM. Oxidative stress refers to the imbalancebetween the production of reactive oxygen species (ROS) and reactivenitrogen species (RNS) and the elimination thereof by the antioxidantdefense system in the body, resulting in excessive production of ROS andRNS, thereby causing damages to histocytes and biologicalmacromolecules, such as proteins and nucleic acids, in the body^([13]).Hyperglycemia is the main cause of oxidative stress, and increases thecontent of ROS and RNS in the body via pathways such as a mitochondrialelectron transport chain^([14]), glucose autooxidation and a polyolpathway^([15]), wherein the mitochondrial electron transport chain isthe predominant pathway of producing ROS. The mitochondrial electrontransport chain mainly involves enzyme complexes I-IV, cytochrome c andcoenzyme Q, wherein a small amount of superoxide products, comprisingsuperoxide anion, hydrogen peroxide and hydroxyl radicals, arecontinuously produced in enzyme complexes I and III, while superoxidedismutase, catalase and glutathione peroxidase catalyze the conversionof superoxide products to oxygen gas and water. However, under obesityor hyperglycemia conditions, the superoxide products are greatlyincreased, and oxidative stress is generated when the rate of productionof the superoxide products exceeds the rate of elimination thereof.

A number of studies^([16-18]) have shown that ROS can directly damagethe β cells, especially destroy cell mitochondrial structure and promoteβ cell apoptosis; ROS may also indirectly inhibit the function of the βcells by affecting the insulin signal transduction pathway, for example,by activating the nuclear transcription factor κB (NF-κB) signal pathwayto cause a β-cell inflammatory response, inhibiting thenucleo-cytoplasmic translocation of pancreatic and duodenal homeobox 1(PDX-1), inhibiting mitochondrial energy metabolism, reducing insulinsynthesis and secretion, etc. Oxidative stress causes a β cell injuryvia the NF-κB pathway, wherein NF-κB is a dimer composed of twosubunits, p50 and RelA, and in a resting cell, it binds to inhibitoryprotein IκB to exist as an inactive trimer in the cytoplasm, which ismainly involved in the response of the cell to stimulations such asstress, cytokines, free radicals, bacteria and viruses, and in thetransient regulation of gene expression, etc.^([19]) Studies have shownthat hyperglycemia-induced ROS activates NF-κB by disruptingintracellular signal transduction and induces β cell injuries^([20]).Mariappan et al.^([21]) inhibited the expression of NF-κB in obese db/dbmice by using pyrrolidine dithiocarbamate (PDTC), and found that thedegree of damage caused by oxidative stress to mitochondria of β cellsin the mice was remarkably reduced; Hofmann et al.^([22]) treateddiabetic patients with anti-oxidant drug α-lipoic acid and found thatthe activity of NF-κB was significantly reduced in the bodies of thepatients, and the condition of the patients was also improved; and Eldoret al.^([23]) specifically inhibited the expression of NF-κB in mice byusing a transgenic technique, which remarkably reduced the incidence ofdiabetes mellitus in the mice induced by STZ.

As a multi-directional nuclear transcription factor, NF-κB is involvedin various gene regulations after being activated, such as cellproliferation, apoptosis, inflammation and immunity^([24]). In a bodywith diabetes mellitus, NF-κB causes leukocytosis of pancreatic islet byregulating the expression of genes of cytokines and chemokines, such asIL-1 (interleukin-1) and MCP-1 (monocyte/macrophage chemoattractantprotein-1) factors, thereby causing a 13 cells injury^([25]). Inaddition, many gene products regulated by NF-κB, such as tumor necrosisfactor α (TNF-α), further activate NF-κB, which aggravates the β cellinjury^([26]).

Studies by Mahadev et al.^([27]) showed that ROS has a regulatory effecton insulin signal transduction, and this effect is versatile. Underinsulin stimulation, the body rapidly produces a trace amount of ROS bymeans of a Nox (NADPH oxidase)-dependent mechanism; the ROS acts as asecond messenger, which mainly inhibits the activity of PTP1B by meansof oxidation to promote an insulin cascade reaction^([28]); furthermore,after Nox is inhibited using DPI (diphenyleneiodonium), thephosphorylation of insulin-stimulated insulin receptor (InsR) andinsulin receptor substrate (IRS) is decreased by 48%^([29]). Studies byLoh et al.^([30]) showed that physiological ROS can promote thesensitivity of the body to insulin. Although in a physiological state, atrace amount of ROS produced by insulin stimulation promotes the actionof insulin, long-term hyperglycemia causes the body to produce a largeamount of ROS via the mitochondrial pathway^([31]), causing insulinresistance.

InsR and IRS are important signaling elements in the insulin signaltransduction pathway: the former is an initiating element for insulinsignal transduction, and the IRS is a bridge between the former and adownstream element in the pathway. Numerous studies have shown thatoxidative stress may interfere with the phosphorylation of InsR and IRSvia multiple pathways to hinder the insulin signal transduction. IKK isan activator for inhibitory subunit IκB of NF-κB, and under ROSstimulation, IKK may act as a kinase for the phosphorylation ofserine/threonine of InsR and IRS, which promotes serine phosphorylationin InsR and IRS, causing normal tyrosine phosphorylation to beinhibited, thereby hindering the insulin signal transduction^([32]).Studies by Brownlee^([33]) showed that IKK can directly phosphorylate aserine residue at site 307 of IRS, resulting in the normal tyrosinephosphorylation of IRS to be reduced, which hinders the binding of InsRto IRS, thereby causing insulin resistance.

In addition to IKK, several members of the MAPK family also have aneffect on InsR and IRS. JNK, extracellular regulated protein kinases(ERK) and p38 mitogen-activated protein kinase (p38 MAPK) are members ofthe MAPK family, have serine/threonine protein kinase activities, andcan be activated under the actions of oxidative stress, cytokines,G-protein coupled receptor agonists, etc. Multiple studies have shownthat the activation of JNK, ERK and p38 MAPK aggravates the degree ofphosphorylation of serine/threonine in InsR and IRS, and the proteinbinding capacity between InsR and IRS and the ability of IRS to activatea downstream signaling molecules containing an SH-2 domain arereduced^([34-36]).

Oxidative stress caused by a diabetic high glucose condition is one ofthe key causes of the formation of various chronic complications, and isalso an important factor in inducing DNA damage^([37]). In case ofdiabetes mellitus, the extracellular fluid has continuous high glucose.In this state, electrons generated by the mitochondrial electrontransport chain are remarkably increased, resulting in excessive ROS,causing damages to the intracellular environment and biologicalmacromolecules such as lipids, proteins, and DNA. Reactive oxygenproduced by the body in the aerobic metabolic pathway acts as amutation-inducing agent to oxidize guanine on the DNA strand to8-hydroxy-2′-deoxyguanosine (8-OHdG). During DNA replication, 8-OHdG isprone to mismatch with adenine, resulting in a G:C to T:A transversionmutation that forms DNA damage. In addition, ROS may further cause otherforms of DNA damage, comprising DNA strand breaks, DNA site mutations,DNA double-strand aberrations, protooncogene and tumor suppressor genemutations, and the like. Furthermore, the DNA damage may also aggravateROS and oxidative stress processes, for example, the DNA damage mayinduce ROS production by means of H2AX-reduced coenzyme II oxidase 1(Nox1)/Rac 1 pathway. ROS further promotes the entry of a large amountof Ca²⁺ into mitochondria, causing cell necrosis and apoptosis, ordirectly damaging mitochondria to cause mitochondrial dysfunction,thereby impairing pancreatic islet β cells and aggravating thepathological process of diabetes mellitus^([38]).

In addition to causing insulin resistance, ROS also has an effect on theinjury of pancreatic islet β cells, and under oxidative stress, theexpression of insulin gene transcription factors, and insulin bindingsites are remarkably reduced, thereby affecting the production andsecretion of insulin. Other adipocytokines such as TNF-α may also reducethe function of the β cells^([15]). The combined action of thesecytokines causes more remarkable damage to the function of thepancreatic islet β cells. In addition, some inflammatory factors mayalso act on the key part of insulin receptor substrate 2 tophosphorylate serine/threonine, which results in accelerated degradationof insulin receptor substrate 2 and promotes apoptosis of pancreaticislet β cells.

It can be seen from the above that the role of oxidative stress in theoccurrence and development of diabetes mellitus is very complicated. Inaddition to directly impairing islet β cells, ROS can also act as asignaling molecule to activate some stress-sensitive pathways, therebyregulating the expression of related factors, causing apoptosis ornecrosis of β cells, inhibiting insulin secretion, inducing insulinresistance, and ultimately causing or aggravating diabetes mellitus.

Treatment of DM

Diabetes mellitus is usually treated by means of medications, andtraditional medications comprise insulin-based drugs and oralhypoglycemic drugs.

In the early days, insulin was mainly extracted from the pancreas ofanimals such as pigs and cattle, and after application to human,remarkable allergic reactions occurred. With increased maturity in the1990s, insulin analogues were gradually applied, and such insulin canremarkably change the pharmacokinetics of traditional insulin, and hasthe advantages of a low incidence of hypoglycemia, fast onset,long-lasting effect, etc. At present, with the deepening of theexploration of insulin preparations, some oral insulin preparations haveentered a testing stage; however, due to technical difficulties, noeffective oral preparations have been applied yet clinically.

There are many traditional oral hypoglycemic drugs, among which thefollowing types are common: (1) biguanides such as metformin. Metforminhas a good cardiovascular protective effect and also a good hypoglycemiceffect, and it has been used as a first-line drug for treating T2DM inmany countries. (2) Sulfonylureas: sulfonylureas are insulinsecretagogues that stimulate pancreatic islet β cells to secreteinsulin, thus achieving an effect of improving the blood glucose level.At present, such insulins that are allowed to be marketed in Chinamainly comprise glimepiride, glibenclamide, glipizide, gliclazide,gliquidone, etc.; however, some studies have shown that if such drugsare taken for a long term, failed hypoglycemic effect may be caused,which easily results in complications such as hypoglycemia and increasedbody mass. (3) Thiazolidinedione compounds (TZD): In 1999, the FDAapproved the use of rosiglitazone and pioglitazone for T2DM, wherein theformer may aggravate the risk of heart diseases and for this reason, itwas later restricted to be used as a second-line treatment drug andprohibited for use in heart failure conditions. In June 2013, the FDAre-examined rosiglitazone, stated that this drug can continue to be usedclinically, and even relaxed or completely unbanned the prohibition ofthe use of this drug and compound preparations thereof. (4)α-glycosidase inhibitors: Such insulins inhibit glycosidase in smallintestinal mucosal epithelial cells, thereby alleviating the absorptionof carbohydrates and leading to a decrease in the postprandial bloodglucose level. Commonly used such drugs comprise voglibose, acarbose,miglitol etc.

At the present stage, drugs for treating diabetes mellitus mainlycomprise traditional antidiabetic drugs, comprising sulfonylureas,glinides, biguanides, thiazolidinediones (TZDs), α-glucosidaseinhibitors, insulin, etc.; however, these drugs all have differentdegrees of adverse reactions, such as triggering hypoglycemia,gastrointestinal discomfort, and obesity. With the deepening of thestudy on the basic theory of diabetes mellitus, people are activelylooking for new therapeutic targets for diabetes mellitus in order toavoid the side effects of traditional hypoglycemic drugs and protect thepancreatic islet β cells. Targets currently found to be associated withthe pathogenesis of diabetes mellitus mainly comprise glucagon-likepeptide-1 (GLP-1), and dipeptidyl peptidase-4 (DPP-4), sodium-glucosecotransporter-2 (SGLT-2), glycogen synthase kinase-3 (GSK-3), proteintyrosine phosphatase (PTP), glucokinase (GK), etc. Among them, glucagonregulation-based drugs such as glucagon-like peptide-1 (GLP-1)analogues, GLP-1 receptor agonists, and dipeptidyl peptidase-4 (DPP-4)inhibitors are considered to be effective in maintaining blood glucosehomeostasis, improving β cell functions, delaying the progression ofdiabetes mellitus, and even reversing the course of diabetes mellitus.

Currently, there is no effective drug or means for completely curingdiabetes mellitus, and current medications focus on reducing anddelaying the occurrence of complications by controlling blood glucosewithin a certain range. With a deeper and more comprehensiveunderstanding of the pathogenesis of diabetes, the study of therapeuticdrugs for diabetes mellitus has also been shifted from the study ofdrugs with traditional mechanisms to the study of drugs with new targetsand new mechanisms of action, wherein some of them have already been onthe market, for example, GLP-1 receptor agonists, DPP-4 inhibitors andSGLT-2 inhibitors, and there are also some drugs in the clinical orpreclinical study stage, e.g. GPR119 receptor agonists, 11β-HSD1inhibitors, PTP1B inhibitors and GK agonists, with the efficacy andsafety having yet to be further clinically verified. Although theemergence of new target-based anti-diabetic drugs in recent years hasprovided more options for DM treatment, since the pathogenesis ofdiabetes mellitus is complex, and a large number of hormones, enzymesand receptors are involved, there are still problems, e.g. single-targetdrugs having a narrow range of action, a weak hypoglycemic effect andcausing adverse reactions after acting on the systemic system, in theresearch field of new drugs, and all of these need to be furtherstudied. Therefore, people need to find more effective therapeutic drugsthat can act on many aspects of the pathogenesis of diabetes mellitus.

The present invention discovers that plasminogen can alleviate thepancreatic tissue injury, control inflammation, reduce pancreatic isletβ cell apoptosis, repair pancreatic tissue, restore the secretionfunction of pancreatic islet β-cells, and reducing blood glucose indiabetic experimental mice, and is expected to become a brand new drugthat comprehensively addresses many aspects of the pathogenesis ofdiabetes mellitus.

BRIEF DESCRIPTION OF THE INVENTION

The present invention comprises the following items:

1. A method for reducing secretion of glucagon in a diabetic subject,comprising administering an effective amount of plasminogen to thesubject.

2. The method of item 1, wherein the plasminogen further reducesexpression of glucagon in the diabetic subject.

3. The method of item 1 or 2, wherein the diabetes mellitus is T1DM orT2DM.

4. The method of any one of items 1 to 3, wherein the plasminogenreduces secretion of glucagon in the diabetic subject after eating.

5. The method of any one of items 1 to 4, wherein the plasminogenreduces secretion of glucagon in the diabetic subject in a fasted state.

6. The method of any one of items 1 to 5, wherein the plasminogenreturns blood glucose to a normal or nearly normal level by reducingsecretion of glucagon in the diabetic subject in an elevated bloodglucose state.

7. The method of any one of items 1 to 6, wherein the plasminogenpromotes expression and/or secretion of insulin while reducing theexpression and/or secretion of glucagon in the subject.

8. The method of item 7, wherein the plasminogen achieves a return to anormal or nearly normal level of blood glucose in the subject bypromoting the expression and/or secretion of insulin while reducingexpression and/or secretion of glucagon in the subject.

9. The method of any of items 1 to 8, wherein the plasminogen promotesexpression of insulin receptor substrate 2 (IRS-2).

10. A method for promoting secretion of insulin in a diabetic subject,comprising administering an effective amount of plasminogen to thesubject.

11. The method of item 10, wherein the plasminogen further promotesexpression of insulin in the diabetic subject.

12. The method of item 10 or 11, wherein the diabetes mellitus is T1DMor T2DM.

13. The method of any one of items 10 to 12, wherein the plasminogenpromotes secretion of insulin in the diabetic subject after eating.

14. The method of any one of items 10 to 12, wherein the plasminogenpromotes secretion of insulin in the diabetic subject in a fasted state.

15. The method of any one of items 10 to 14, wherein the plasminogenreturns blood glucose to a normal or nearly normal level by promotingsecretion of insulin in response to a stimulation of elevated bloodglucose in the diabetic subject.

16. The method of any one of items 10 to 15, wherein the plasminogenreduces expression and/or secretion of glucagon in the subject whilepromoting the expression and/or secretion of insulin.

17. The method of any one of item 16, wherein the plasminogen achieves areturn to a normal or nearly normal level of blood glucose in thesubject by reducing expression and/or secretion of glucagon in thesubject while promoting the expression and/or secretion of insulin.

18. A method for reducing blood glucose in a diabetic subject,comprising administering an effective amount of plasminogen to thesubject.

19. The method of item 18, wherein the blood glucose is selected fromone or more of: a serum glucose level, a serum fructosamine level, and aserum glycated hemoglobin level.

20. The method of item 19, wherein the blood glucose is a serum glucoselevel.

21. The method of any one of items 18 to 20, wherein the diabetesmellitus is T1DM or T2DM.

22. A method for improving the glucose tolerance in a diabetic subject,comprising administering an effective amount of plasminogen to thesubject.

23. The method of item 22, wherein the diabetes mellitus is T2DM.

24. A method for promoting postprandial blood glucose drop in a diabeticsubject, comprising administering an effective amount of plasminogen tothe subject.

25. The method of item 24, wherein the plasminogen is administered 30minutes to 1.5 hour before the subject has a meal.

26. The method of item 25, wherein the plasminogen is administered 30minutes to 1 hour before the subject has a meal.

27. A method for promoting the utilization of glucose in a diabeticsubject, comprising administering an effective amount of plasminogen tothe subject.

28. The method of any one of items 1 to 27, wherein the plasminogen isadministered in combination with one or more other drugs or therapies.

29. The method of item 28, wherein the plasminogen is administered incombination with one or more drugs selected from anti-diabetic drugs,drugs against cardiovascular and cerebrovascular diseases,anti-thrombotic drugs, anti-hypertensive drugs, antilipemic drugs,anticoagulant drugs, and anti-infective drugs.

30. The method of any one of items 1 to 29, wherein the plasminogen hasat least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identitywith SEQ ID No. 2, 6, 8, 10 or 12, and still has the plasminogenactivity.

31. The method of any one of items 1 to 30, wherein the plasminogen is aprotein that has 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-45, 1-40, 1-35,1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2 or 1 amino acid added,deleted and/or substituted in SEQ ID No. 2, 6, 8, 10 or 12, and stillhas the plasminogen activity.

32. The method of any one of items 1 to 31, wherein the plasminogen is aprotein that comprises a plasminogen active fragment and still has theplasminogen activity.

33. The method of any one of items 1 to 32, wherein the plasminogen isselected from Glu-plasminogen, Lys-plasminogen, mini-plasminogen,micro-plasminogen, delta-plasminogen or their variants that retain theplasminogen activity.

34. The method of any one of items 1 to 33, wherein the plasminogen is anatural or synthetic human plasminogen, or a variant or fragment thereofthat still retains the plasminogen activity.

35. The method of any one of items 1 to 33, wherein the plasminogen isan ortholog of human plasminogen from a primate or a rodent, or avariant or fragment thereof that still retains the plasminogen activity.

36. The method of any one of items 1 to 35, wherein the amino acids ofthe plasminogen are as shown in SEQ ID No. 2, 6, 8, 10 or 12.

37. The method of any one of items 1 to 36, wherein the plasminogen is anatural human plasminogen.

38. The method of any one of items 1 to 37, wherein the subject is ahuman.

39. The method of any one of items 1 to 38, wherein the subject has alack or deficiency of plasminogen.

40. The method of any one of items 1 to 39, wherein the lack ordeficiency is congenital, secondary and/or local.

41. A plasminogen for use in the method of any one of items 1 to 40.

42. A pharmaceutical composition, comprising a pharmaceuticallyacceptable carrier and the plasminogen for use in the method of any oneof items 1 to 40.

43. A preventive or therapeutic kit comprising: (i) the plasminogen foruse in the method of any one of items 1 to 40, and (ii) a means fordelivering the plasminogen to the subject.

44. The kit of item 43, wherein the means is a syringe or a vial.

45. The kit of item 43 or 44, further comprising a label or aninstruction for use indicating the administration of the plasminogen tothe subject to implement the method of any one of items 1 to 40.

46. An article of manufacture, comprising:

a container comprising a label; and

(i) the plasminogen for use in the method of any one of items 1 to 40 ora pharmaceutical composition comprising the plasminogen, wherein thelabel indicates the administration of the plasminogen or the compositionto the subject to implement the method of any one of items 1 to 40.

47. The kit of any one of items 43 to 45 or the article of manufactureof item 46, further comprising one or more additional means orcontainers containing other drugs.

48. The kit or the article of manufacture of item 47, wherein the otherdrugs are selected from the group of anti-diabetic drugs, drugs againstcardiovascular and cerebrovascular diseases, anti-thrombotic drugs,anti-hypertensive drugs, antilipemic drugs, anticoagulant drugs, andanti-infective drugs.

In one aspect, the present invention relates to a method for preventingand/or treating diabetes mellitus, comprising administering atherapeutically effective amount of plasminogen or plasmin to a subject.

In another aspect, the present invention relates to a method forreducing blood glucose in a diabetic subject, comprising administeringan effective amount of plasminogen to the subject. The present inventionfurther relates to the use of plasminogen for reducing blood glucose ina diabetic subject. The present invention further relates to the use ofplasminogen in the preparation of a medicament for reducing bloodglucose in a diabetic subject. In addition, the present inventionfurther relates to a plasminogen for reducing blood glucose in adiabetic subject. In some embodiments, the blood glucose is selectedfrom one or more of: a serum glucose level, a serum fructosamine level,and a serum glycated hemoglobin level. In some other embodiments, theblood glucose is a serum glucose level. In the above-mentionedembodiments, the diabetes mellitus is T1DM or T2DM.

In another aspect, the present invention relates to a method forimproving the glucose tolerance in a diabetic subject, comprisingadministering an effective amount of plasminogen to the subject. Thepresent invention further relates to the use of plasminogen forincreasing glucose tolerance in a diabetic subject. The presentinvention further relates to the use of plasminogen in the preparationof a medicament for increasing glucose tolerance in a diabetic subject.In addition, the present invention further relates to a plasminogen forincreasing glucose tolerance in a diabetic subject. In some embodiments,the diabetes mellitus is T2DM.

In one aspect, the present invention relates to a method for promotingpostprandial blood glucose drop in a diabetic subject, comprisingadministering an effective amount of plasminogen to the subject. Thepresent invention further relates to the use of plasminogen forpromoting postprandial blood glucose drop in a diabetic subject. Thepresent invention further relates to the use of plasminogen in thepreparation of a medicament for promoting postprandial blood glucosedrop in a diabetic subject. In addition, the present invention furtherrelates to a plasminogen for promoting postprandial blood glucose dropin a diabetic subject. In some embodiments, the plasminogen isadministered 30 minutes to 1.5 hours before the subject has a meal. Insome other embodiments, the plasminogen is administered 30 minutes to 1hour before the subject has a meal.

In one aspect, the present invention relates to a method for promotingthe utilization of glucose in a diabetic subject, comprisingadministering an effective amount of plasminogen to the subject. Thepresent invention further relates to the use of plasminogen forpromoting the utilization of glucose in a diabetic subject. The presentinvention further relates to the use of plasminogen in the preparationof a medicament for promoting the utilization of glucose in a diabeticsubject. In addition, the present invention further relates to aplasminogen for promoting the utilization of glucose in a diabeticsubject. In another aspect, the present invention relates to a methodfor promoting secretion of insulin in a diabetic subject, comprisingadministering an effective amount of plasminogen to the subject. Inother embodiments, the plasminogen further promotes the expression ofinsulin in a diabetic subject. In the above-mentioned embodiments, thediabetes mellitus is T1DM or T2DM. In some embodiments, the plasminogenpromotes secretion of insulin in the diabetic subject after eating. Insome other embodiments, the plasminogen promotes secretion of insulin inthe diabetic subject in a fasted state. In some embodiments, theplasminogen returns blood glucose to a normal or nearly normal level bypromoting secretion of insulin in response to an elevated blood glucosestimulation in the diabetic subject. In some other embodiments, theplasminogen reduces expression and/or secretion of glucagon in thesubject while promoting the expression and/or secretion of insulin; inparticular, the plasminogen achieves a return to a normal or nearlynormal level of blood glucose in the subject by reducing expressionand/or secretion of glucagon in the subject while promoting theexpression and/or secretion of insulin.

In one aspect, the present invention relates to a method for reducingsecretion of glucagon in a diabetic subject, comprising administering aneffective amount of plasminogen to the subject. The present inventionfurther relates to the use of plasminogen for reducing secretion ofglucagon in a diabetic subject. The present invention further relates tothe use of plasminogen in the preparation of a medicament for reducingsecretion of glucagon in a diabetic subject. In addition, the presentinvention further relates to a plasminogen for reducing secretion ofglucagon in a diabetic subject. In some embodiments, the plasminogenfurther reduces expression of glucagon in the diabetic subject. In theabove-mentioned embodiments, the diabetes mellitus is T1DM or T2DM. Insome embodiments, the plasminogen reduces secretion of glucagon in thediabetic subject after eating. In some other embodiments, theplasminogen reduces secretion of glucagon in the diabetic subject in afasted state. In some embodiments, the plasminogen returns blood glucoseto a normal or nearly normal level by reducing secretion of glucagon inthe diabetic subject in an elevated blood glucose state. In someembodiments, the plasminogen returns blood glucose to a normal or nearlynormal level by reducing secretion of glucagon in the diabetic subjectin an elevated blood glucose state. In some other embodiments, theplasminogen promotes expression and/or secretion of insulin whilereducing the expression and/or secretion of glucagon in the subject; inparticular, the plasminogen achieves a return to a normal or nearlynormal level of blood glucose in the subject by promoting the expressionand/or secretion of insulin while reducing expression and/or secretionof glucagon in the subject. In the above-mentioned embodiments, theplasminogen promotes expression of insulin receptor substrate 2 (IRS-2).

In one aspect, the present invention relates to a method for promotingrepair of a pancreatic islet cell injury in a diabetic subject,comprising administering an effective amount of plasminogen to thesubject. The present invention further relates to the use of plasminogenfor promoting repair of a pancreatic islet cell injury in a diabeticsubject. The present invention further relates to the use of plasminogenin the preparation of a medicament for promoting repair of a pancreaticislet cell injury in a diabetic subject. In addition, the presentinvention further relates to a plasminogen for promoting repair of apancreatic islet cell injury in a diabetic subject. In some embodiments,the plasminogen promotes expression of insulin receptor substrate 2(IRS-2). In some other embodiments, the plasminogen promotes expressionof cytokine TNF-α. In some other embodiments, the plasminogen promotesexpression of multi-directional nuclear transcription factor NF-κB inthe subject. In some embodiments, the pancreatic islet cell injury isone or more selected from: an injured insulin synthesis and secretionfunction of pancreatic islet β cells, an injured pancreatic islet tissuestructure, collagen deposition in the pancreatic islet, pancreatic isletfibrosis, pancreatic islet cell apoptosis, a disordered balance betweenthe secretion of glucagon and of insulin in the pancreatic islet, andfailed adaptation of levels of glucagon and insulin secreted by thepancreatic islet to a blood glucose level in a subject. In someembodiments, the plasminogen reduces secretion of glucagon and increasessecretion of insulin in the diabetic subject; in particular, the normalbalance between the secretion of glucagon and of insulin in thepancreatic islet is repaired.

In another aspect, the present invention relates to a method forprotecting the pancreatic islet of a subject, comprising administeringan effective amount of plasminogen to the subject. The present inventionfurther relates to the use of plasminogen for protecting the pancreaticislet of a subject. The present invention further relates to the use ofplasminogen in the preparation of a medicament for protecting thepancreatic islet of a subject. In addition, the present inventionfurther relates to a plasminogen for protecting the pancreatic islet ofa subject. In some embodiments, the plasminogen reduces collagendeposition in the pancreatic islet. In some other embodiments, theplasminogen reduces pancreatic islet fibrosis. In some otherembodiments, the plasminogen reduces pancreatic islet cell apoptosis. Insome other embodiments, the plasminogen promotes expression of insulinreceptor substrate 2 (IRS-2) in the pancreatic islet. In someembodiments, the plasminogen promotes repair of an inflammation in thepancreatic islet. In some other embodiments, the plasminogen promotesexpression of cytokine TNF-α. In some other embodiments, the plasminogenpromotes expression of multi-directional nuclear transcription factorNF-κB in the subject. In the above-mentioned embodiments, the subject isa diabetic patient; in particular, the diabetic patient has T1DM orT2DM. In some embodiments, the subject with T1DM is a subject withnormal PLG activity or impaired PLG activity.

In one aspect, the present invention relates to a method for promotingrepair of an inflammation in the pancreatic islet, comprisingadministering an effective amount of plasminogen to the subject. Thepresent invention further relates to the use of plasminogen forpromoting repair of an inflammation in the pancreatic islet of adiabetic subject. The present invention further relates to the use ofplasminogen in the preparation of a medicament for promoting repair ofan inflammation in the pancreatic islet of a diabetic subject. Inaddition, the present invention further relates to a plasminogen forpromoting repair of an inflammation in the pancreatic islet of adiabetic subject. In some embodiments, the plasminogen promotesexpression of cytokine TNF-α. In some other embodiments, the plasminogenpromotes expression of multi-directional nuclear transcription factorNF-κB in the subject. In some other embodiments, the plasminogen reducescollagen deposition in the pancreatic islet. In some other embodiments,the plasminogen reduces pancreatic islet fibrosis. In some otherembodiments, the plasminogen inhibits pancreatic islet cell apoptosis.In the above-mentioned embodiments, the diabetic patient has T1DM orT2DM; in particular, the subject with T1DM is a subject with normal PLGactivity or impaired PLG activity.

In one aspect, the present invention relates to a method for promotingexpression of cytokine TNF-α in a diabetic subject, comprisingadministering an effective amount of plasminogen to the subject. Thepresent invention further relates to the use of plasminogen forpromoting expression of cytokine TNF-α in a diabetic subject. Thepresent invention further relates to the use of plasminogen in thepreparation of a medicament for promoting expression of cytokine TNF-αin a diabetic subject. In addition, the present invention furtherrelates to a plasminogen for promoting expression of cytokine TNF-α in adiabetic subject.

In another aspect, the present invention relates to a method forpromoting expression of multi-directional nuclear transcription factorNF-κB in a diabetic subject, comprising administering an effectiveamount of plasminogen to the subject. The present invention furtherrelates to the use of plasminogen for promoting expression ofmulti-directional nuclear transcription factor NF-κB in a diabeticsubject. The present invention further relates to the use of plasminogenin the preparation of a medicament for promoting expression ofmulti-directional nuclear transcription factor NF-κB in a diabeticsubject.

In another aspect, the present invention relates to a method forpromoting expression of insulin receptor substrate 2 (IRS-2) by thepancreatic islet, comprising administering an effective amount ofplasminogen to the subject. The present invention further relates to theuse of plasminogen for promoting expression of insulin receptorsubstrate 2 (IRS-2) in the pancreatic islet. The present inventionfurther relates to the use of plasminogen in the preparation of amedicament for promoting expression of insulin receptor substrate 2(IRS-2) in the pancreatic islet. In addition, the present inventionfurther relates to a plasminogen for promoting expression of insulinreceptor substrate 2 (IRS-2) in the pancreatic islet.

In another aspect, the present invention relates to a method forpromoting secretion of insulin in a diabetic subject, comprisingadministering an effective amount of plasminogen to the subject topromote expression of insulin receptor substrate 2 (IRS-2). The presentinvention further relates to the use of plasminogen for promotingsecretion of insulin in a diabetic subject. The present inventionfurther relates to the use of plasminogen in the preparation of amedicament for promoting secretion of insulin in a diabetic subject. Inaddition, the present invention further relates to a plasminogen forpromoting secretion of insulin in a diabetic subject.

In another aspect, the present invention relates to a method forpromoting an increase in the number of pancreatic islet β cells in adiabetic subject, comprising administering an effective amount ofplasminogen to the subject. The present invention further relates to theuse of plasminogen for promoting an increase in the number of pancreaticislet β cells in a diabetic subject. The present invention furtherrelates to the use of plasminogen in the preparation of a medicament forpromoting an increase in the number of pancreatic islet β cells in adiabetic subject. In addition, the present invention further relates toa plasminogen for promoting an increase in the number of pancreaticislet β cells in a diabetic subject. In some embodiments, theplasminogen promotes expression of insulin receptor substrate 2 (IRS-2).

In one aspect, the present invention relates to a method for reducingpancreatic islet β cell apoptosis, comprising administering an effectiveamount of plasminogen to a subject. The present invention furtherrelates to the use of plasminogen for reducing pancreatic islet β cellapoptosis. The present invention further relates to the use ofplasminogen in the preparation of a medicament for reducing pancreaticislet β cell apoptosis. In addition, the present invention furtherrelates to a plasminogen for reducing pancreatic islet β cell apoptosis.In some embodiments, the plasminogen promotes expression of insulinreceptor substrate 2 (IRS-2).

In another aspect, the present invention relates to a method forpromoting repair of a pancreatic islet β cell injury, comprisingadministering an effective amount of plasminogen to a subject. Thepresent invention further relates to the use of plasminogen forpromoting repair of a pancreatic islet β cell injury. The presentinvention further relates to the use of plasminogen in the preparationof a medicament for promoting repair of a pancreatic islet β cellinjury. The present invention further relates to a plasminogen forpromoting repair of a pancreatic islet β cell injury. In someembodiments, the plasminogen promotes expression of insulin receptorsubstrate 2 (IRS-2).

In another aspect, the present invention relates to a method forpromoting recovery of pancreatic islet β cell function, comprisingadministering an effective amount of plasminogen to a subject. Thepresent invention further relates to the use of plasminogen forpromoting recovery of pancreatic islet β cell function. The presentinvention further relates to the use of plasminogen in the preparationof a medicament for promoting recovery of pancreatic islet β cellfunction. In addition, the present invention further relates to aplasminogen for promoting recovery of pancreatic islet β cell function.In some embodiments, the plasminogen promotes expression of insulinreceptor substrate 2 (IRS-2).

In the above-mentioned embodiments, the plasminogen is administered incombination with one or more other drugs or therapies. In particular,the plasminogen may be administered in combination with one or moredrugs selected from anti-diabetic drugs, drugs against cardiovascularand cerebrovascular diseases, anti-thrombotic drugs, anti-hypertensivedrugs, antilipemic drugs, anticoagulant drugs, and anti-infective drugs.

In the above-mentioned embodiments, the plasminogen has at least 75%,80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ IDNo. 2, 6, 8, 10 or 12, and still has the activity of plasminogen.

In the above-mentioned embodiments, the amino acids of the plasminogenare as shown in SEQ ID No. 2, 6, 8, 10 or 12. In some embodiments, theplasminogen is a protein that has 1-100, 1-90, 1-80, 1-70, 1-60, 1-50,1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-4, 1-3, 1-2 or 1amino acid added, deleted and/or substituted in SEQ ID No. 2, 6, 8, 10or 12, and still has the activity of plasminogen.

In the above-mentioned embodiments, the plasminogen is a protein thatcomprises a plasminogen active fragment and still has the activity ofplasminogen. Specifically, the plasminogen is selected fromGlu-plasminogen, Lys-plasminogen, mini-plasminogen, micro-plasminogen,delta-plasminogen or their variants that retain the plasminogenactivity.

In the above-mentioned embodiments, the plasminogen is a natural orsynthetic human plasminogen, or a variant or fragment thereof that stillretains the plasminogen activity. In some embodiments, the plasminogenis an ortholog of human plasminogen from a primate or a rodent, or avariant or fragment thereof that still retains the plasminogen activity.For example, the plasminogen is an ortholog of plasminogen from primatesor rodents, for example, an ortholog of plasminogen from gorillas,rhesus monkeys, murine, cows, horses and dogs. Most preferably, theamino acid sequence of the plasminogen of the present invention is asshown in SEQ ID No. 2, 6, 8, 10 or 12.

In the above-mentioned embodiments, the subject is a human. In someembodiments, the subject has a lack or deficiency of plasminogen.Specifically, the lack or deficiency is congenital, secondary and/orlocal.

In one embodiment, the plasminogen is administered by systemic ortopical route, preferably by the following routes: topical, intravenous,intramuscular, subcutaneous, inhalation, intraspinal, local injection,intraarticular injection or rectal route. In one embodiment, the topicaladministration is performed by direct administration to osteoporoticareas, for example through a means such as a dressing and a catheter.

In one embodiment, the plasminogen is administered in combination with asuitable polypeptide carrier or stabilizer. In one embodiment, theplasminogen is administered at a dosage of 0.0001-2000 mg/kg, 0.001-800mg/kg, 0.01-600 mg/kg, 0.1-400 mg/kg, 1-200 mg/kg, 1-100 mg/kg or 10-100mg/kg (by per kg of body weight) or 0.0001-2000 mg/cm², 0.001-800mg/cm², 0.01-600 mg/cm², 0.1-400 mg/cm², 1-200 mg/cm², 1-100 mg/cm² or10-100 mg/cm² (by per square centimeter of body surface area) daily,preferably the dosage is repeated at least once, preferably the dosageis administered at least daily. In the case of local administration, theabove dosages may also be further adjusted depending on thecircumstances. In one aspect, the present invention relates to apharmaceutical composition, comprising a pharmaceutically acceptablecarrier and the plasminogen for use in the method of the presentinvention.

In another aspect, the present invention relates to a preventive ortherapeutic kit comprising: (i) the plasminogen for use in the method ofthe present invention, and (ii) a means for delivering the plasminogento the subject, in particular, the means is a syringe or a vial. In someembodiments, the kit further comprises a label or an instruction for useindicating the administration of the plasminogen to the subject toimplement the methods of the present invention.

In another aspect, the present invention further relates to an articleof manufacture comprising: a container comprising a label; and (i) theplasminogen for use in the methods of the present invention or apharmaceutical composition comprising the plasminogen, wherein the labelindicates the administration of the plasminogen or the composition tothe subject to implement the methods of the present invention.

In the above-mentioned embodiments, the kit or the article ofmanufacture further comprises one or more additional means or containerscontaining other drugs. In some embodiments, the other drugs areselected from the group of anti-diabetic drugs, drugs againstcardiovascular and cerebrovascular diseases, anti-thrombotic drugs,anti-hypertensive drugs, antilipemic drugs, anticoagulant drugs, andanti-infective drugs.

DETAILED DESCRIPTION OF EMBODIMENTS

“Diabetes mellitus” is a series of dysmetabolic syndromes ofcarbohydrates, proteins, fats, water, electrolytes and the like that arecaused by islet hypofunction, insulin resistance and the like resultingfrom the effects of genetic factors, immune dysfunction, microbialinfections and toxins thereof, free radical toxins, mental factors andother various pathogenic factors on the body, and is mainlycharacterized by hyperglycemia clinically.

“Diabetic complications” are damages to or dysfunctions of other organsor tissues of the body caused by poor blood glucose control duringdiabetes mellitus, including damages to or dysfunctions of the liver,kidneys, heart, retina, nervous system damage and the like. According tostatistics of the World Health Organization, there are up to more than100 diabetic complications, and diabetes mellitus is a disease currentlyknown to have the most complications.

“Insulin resistance” refers to a decrease in the efficiency of insulinin promoting glucose uptake and utilization for various reasons,resulting in compensatory secretion of excess insulin in the body, whichcauses hyperinsulinemia to maintain blood glucose stability.

“Plasmin” is a very important enzyme that exists in the blood and iscapable of degrading fibrin multimers.

“Plasminogen (plg)” is the zymogen form of plasmin, which is aglycoprotein composed of 810 amino acids calculated based on the aminoacid sequence (SEQ ID No. 4) of the natural human plasminogen containinga signal peptide according to the sequence in the swiss prot, having amolecular weight of about 90 kD, being synthesized mainly in the liverand being capable of circulating in the blood, with the cDNA sequencethat encodes this amino acid sequence is as shown in SEQ ID No. 3.Full-length PLG contains seven domains: a C-terminal serine proteasedomain, an N-terminal Pan Apple (PAp) domain and five Kringle domains(Kringles 1-5). Referring to the sequence in the swiss prot, the signalpeptide comprises residues Met1-Gly19, PAp comprises residuesGlu20-Val98, Kringle 1 comprises residues Cys103-Cys181, Kringle 2comprises residues Glu184-Cys262, Kringle 3 comprises residuesCys275-Cys352, Kringle 4 comprises residues Cys377-Cys454, and Kringle 5comprises residues Cys481-Cys560. According to the NCBI data, the serineprotease domain comprises residues Val581-Arg804.

Glu-plasminogen is a natural full-length plasminogen and is composed of791 amino acids (without a signal peptide of 19 amino acids); the cDNAsequence encoding this sequence is as shown in SEQ ID No. 1; and theamino acid sequence is as shown in SEQ ID No. 2. In vivo,Lys-plasminogen, which is formed by hydrolysis of amino acids atpositions 76-77 of Glu-plasminogen, is also present, as shown in SEQ IDNo. 6; and the cDNA sequence encoding this amino acid sequence is asshown in SEQ ID No. 5. δ-plasminogen is a fragment of full-lengthplasminogen that lacks the structure of Kringle 2-Kringle 5 and containsonly Kringle 1 and the serine protease domain^([39,40]). The amino acidsequence (SEQ ID No. 8) of δ-plasminogen has been reported in theliterature^([40]), and the cDNA sequence encoding this amino acidsequence is as shown in SEQ ID No. 7. Mini-plasminogen is composed ofKringle 5 and the serine protease domain, and has been reported in theliterature to comprise residues Val443-Asn791 (with the Glu residue ofthe Glu-plg sequence that does not contain a signal peptide as thestarting amino acid)^([41]); the amino acid sequence is as shown in SEQID No. 10; and the cDNA sequence encoding this amino acid sequence is asshown in SEQ ID No. 9. In addition, micro-plasminogen comprises only theserine protease domain, the amino acid sequence of which has beenreported in the literature to comprise residues Ala543-Asn791 (with theGlu residue of the Glu-plg sequence that does not contain a signalpeptide as the starting amino acid)^([42]), and the sequence of whichhas been also reported in patent CN 102154253 A to comprise residuesLys531-Asn791 (with the Glu residue of the Glu-plg sequence that doesnot contain a signal peptide as the starting amino acid) (the sequencein this patent application refers to the patent CN 102154253 A); theamino acid sequence is as shown in SEQ ID No. 12; and the cDNA sequenceencoding this amino acid sequence is as shown in SEQ ID No. 11.

In the present invention, “plasmin” is used interchangeably with“fibrinolysin” and “fibrinoclase”, and the terms have the same meaning;and “plasminogen” is used interchangeably with “profibrinolysin” and“fibrinoclase zymogen”, and the terms have the same meaning.

In the present application, the meaning of “lack” in plasminogen is thatthe content or activity of plasminogen in the body of a subject is lowerthan that of a normal person, which is low enough to affect the normalphysiological function of the subject; and the meaning of “deficiency”in plasminogen is that the content or activity of plasminogen in thebody of a subject is significantly lower than that of a normal person,or even the activity or expression is extremely small, and only throughexogenous supply can the normal physiological function be maintained.

Those skilled in the art can understand that all the technical solutionsof the plasminogen of the present invention are suitable for plasmin.Therefore, the technical solutions described in the present inventioncover plasminogen and plasmin.

In the embodiments of the present invention, “aging” and “prematureaging” are used interchangeably to mean the same meaning.

In the course of circulation, plasminogen is in a closed, inactiveconformation, but when bound to thrombi or cell surfaces, it isconverted into an active PLM in an open conformation under the mediationof a PLG activator (plasminogen activator, PA). The active PLM canfurther hydrolyze the fibrin clots to fibrin degradation products andD-dimers, thereby dissolving the thrombi. The PAp domain of PLGcomprises an important determinant that maintains plasminogen in aninactive, closed conformation, and the KR domain is capable of bindingto lysine residues present on receptors and substrates. A variety ofenzymes that can serve as PLG activators are known, including: tissueplasminogen activator (tPA), urokinase plasminogen activator (uPA),kallikrein, coagulation factor XII (Hagmann factor), and the like.

“Plasminogen active fragment” refers to an active fragment in theplasminogen protein that is capable of binding to a target sequence in asubstrate and exerting the proteolytic function. The technical solutionsof the present invention involving plasminogen encompass technicalsolutions in which plasminogen is replaced with a plasminogen activefragment. The plasminogen active fragment of the present invention is aprotein comprising a serine protease domain of plasminogen. Preferably,the plasminogen active fragment of the present invention comprises SEQID No. 14, or an amino acid sequence having an amino acid sequenceidentity of at least 80%, 90%, 95%, 96%, 97%, 98% or 99% with SEQ ID No.14. Therefore, plasminogen of the present invention comprises a proteincontaining the plasminogen active fragment and still having theplasminogen activity.

At present, methods for determining plasminogen and its activity inblood include: detection of tissue plasminogen activator activity(t-PAA), detection of tissue plasminogen activator antigen (t-PAAg) inplasma, detection of tissue plasminogen activity (plgA) in plasma,detection of tissue plasminogen antigen (plgAg) in plasma, detection ofactivity of the inhibitor of tissue plasminogen activators in plasma,detection of inhibitor antigens of tissue plasminogen activators inplasma and detection of plasmin-anti-plasmin (PAP) complex in plasma.The most commonly used detection method is the chromogenic substratemethod: streptokinase (SK) and a chromogenic substrate are added to atest plasma, the PLG in the test plasma is converted into PLM by theaction of SK, PLM acts on the chromogenic substrate, and then it isdetermined that the increase in absorbance is directly proportional toplasminogen activity using a spectrophotometer. In addition, plasminogenactivity in blood can also be determined by immunochemistry, gelelectrophoresis, immunonephelometry, radioimmuno-diffusion and the like.

“Orthologues or orthologs” refer to homologs between different species,including both protein homologs and DNA homologs, and are also known asorthologous homologs and vertical homologs. The term specifically refersto proteins or genes that have evolved from the same ancestral gene indifferent species. The plasminogen of the present invention includeshuman natural plasminogen, and also includes orthologues or orthologs ofplasminogens derived from different species and having plasminogenactivity.

“Conservatively substituted variant” refers to one in which a givenamino acid residue is changed without altering the overall conformationand function of the protein or enzyme, including, but not limited to,replacing an amino acid in the amino acid sequence of the parent proteinby an amino acid with similar properties (such as acidity, alkalinity,hydrophobicity, etc.). Amino acids with similar properties are wellknown. For example, arginine, histidine and lysine are hydrophilic basicamino acids and are interchangeable. Similarly, isoleucine is ahydrophobic amino acid that can be replaced by leucine, methionine orvaline. Therefore, the similarity of two proteins or amino acidsequences with similar functions may be different. For example, thesimilarity (identity) is 70%-99% based on the MEGALIGN algorithm.“Conservatively substituted variant” also includes a polypeptide orenzyme having amino acid identity of 60% or more, preferably 75% ormore, more preferably 85% or more, even more preferably 90% or more asdetermined by the BLAST or FASTA algorithm, and having the same orsubstantially similar properties or functions as the natural or parentprotein or enzyme.

“Isolated” plasminogen refers to the plasminogen protein that isisolated and/or recovered from its natural environment. In someembodiments, the plasminogen will be purified (1) to a purity of greaterthan 90%, greater than 95% or greater than 98% (by weight), asdetermined by the Lowry method, such as more than 99% (by weight); (2)to a degree sufficiently to obtain at least 15 residues of theN-terminal or internal amino acid sequence using a spinning cupsequenator; or (3) to homogeneity, which is determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing ornon-reducing conditions using Coomassie blue or silver staining.Isolated plasminogen also includes plasminogen prepared from recombinantcells by bioengineering techniques and separated by at least onepurification step.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein and refer to polymeric forms of amino acids ofany length, which may include genetically encoded and non-geneticallyencoded amino acids, chemically or biochemically modified or derivatizedamino acids, and polypeptides having modified peptide backbones. Theterm includes fusion proteins, including, but not limited to, fusionproteins having heterologous amino acid sequences, fusions havingheterologous and homologous leader sequences (with or without N-terminalmethionine residues); and the like.

The “percent amino acid sequence identity (%)” with respect to thereference polypeptide sequence is defined as the percentage of aminoacid residues in the candidate sequence identical to the amino acidresidues in the reference polypeptide sequence when a gap is introducedas necessary to achieve maximal percent sequence identity and noconservative substitutions are considered as part of sequence identity.The comparison for purposes of determining percent amino acid sequenceidentity can be achieved in a variety of ways within the skill in theart, for example using publicly available computer softwares, such asBLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled inthe art can determine appropriate parameters for aligning sequences,including any algorithm needed to achieve the maximum comparison overthe full length of the sequences being compared. However, for purposesof the present invention, the percent amino acid sequence identity valueis generated using the sequence comparison computer program ALIGN-2.

In the case of comparing amino acid sequences using ALIGN-2, the % aminoacid sequence identity of a given amino acid sequence A relative to agiven amino acid sequence B (or may be expressed as a given amino acidsequence A having or containing a certain % amino acid sequence identityrelative to, with or for a given amino acid sequence B) is calculated asfollows:fraction X/Y×100

wherein X is the number of identically matched amino acid residuesscored by the sequence alignment program ALIGN-2 in the alignment of Aand B using the program, and wherein Y is the total number of amino acidresidues in B. It will be appreciated that where the length of aminoacid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A relative to B will not be equal tothe % amino acid sequence identity of B relative to A. Unlessspecifically stated otherwise, all the % amino acid sequence identityvalues used herein are obtained using the ALIGN-2 computer program asdescribed in the previous paragraph.

As used herein, the terms “treatment” and “prevention” refer toobtaining a desired pharmacological and/or physiologic effect. Theeffect may be complete or partial prevention of a disease or itssymptoms and/or partial or complete cure of the disease and/or itssymptoms, and includes: (a) prevention of the disease from developing ina subject that may have a predisposition to the disease but has not beendiagnosed as having the disease; (b) suppression of the disease, i.e.,blocking its formation; and (c) alleviation of the disease and/or itssymptoms, i.e., eliminating the disease and/or its symptoms.

The terms “individual”, “subject” and “patient” are used interchangeablyherein and refer to mammals, including, but not limited to, murine (ratsand mice), non-human primates, humans, dogs, cats, hoofed animals (e.g.,horses, cattle, sheep, pigs, goats) and so on.

“Therapeutically effective amount” or “effective amount” refers to anamount of plasminogen sufficient to achieve the prevention and/ortreatment of a disease when administered to a mammal or another subjectto treat the disease. The “therapeutically effective amount” will varydepending on the plasminogen used, the severity of the disease and/orits symptoms, as well as the age, body weight of the subject to betreated, and the like.

2. Preparation of the Plasminogen of the Present Invention

Plasminogen can be isolated and purified from nature for furthertherapeutic uses, and can also be synthesized by standard chemicalpeptide synthesis techniques. When chemically synthesized, a polypeptidecan be subjected to liquid or solid phase synthesis. Solid phasepolypeptide synthesis (SPPS) is a method suitable for chemical synthesisof plasminogen, in which the C-terminal amino acid of a sequence isattached to an insoluble support, followed by the sequential addition ofthe remaining amino acids in the sequence. Various forms of SPPS, suchas Fmoc and Boc, can be used to synthesize plasminogen. Techniques forsolid phase synthesis are described in Barany and Solid-Phase PeptideSynthesis; pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol.2: Special Methods in Peptide Synthesis, Part A., Merrifield, et al. J.Am. Chem. Soc., 85: 2149-2156 (1963); Stewart et al. Solid Phase PeptideSynthesis, 2nd ed. Pierce Chem. Co., Rockford, Ill. (1984); and GanesanA. 2006 Mini Rev. Med Chem. 6:3-10 and Camarero J A et al. 2005 ProteinPept Lett. 12:723-8. Briefly, small insoluble porous beads are treatedwith a functional unit on which a peptide chain is constructed. Afterrepeated cycles of coupling/deprotection, the attached solid phase freeN-terminal amine is coupled to a single N-protected amino acid unit.This unit is then deprotected to expose a new N-terminal amine that canbe attached to another amino acid. The peptide remains immobilized onthe solid phase before it is cut off.

Standard recombinant methods can be used to produce the plasminogen ofthe present invention. For example, a nucleic acid encoding plasminogenis inserted into an expression vector, so that it is operably linked toa regulatory sequence in the expression vector. Expression regulatorysequence includes, but is not limited to, promoters (e.g., naturallyassociated or heterologous promoters), signal sequences, enhancerelements and transcription termination sequences. Expression regulationcan be a eukaryotic promoter system in a vector that is capable oftransforming or transfecting eukaryotic host cells (e.g., COS or CHOcells). Once the vector is incorporated into a suitable host, the hostis maintained under conditions suitable for high-level expression of thenucleotide sequence and collection and purification of plasminogen.

A suitable expression vector is usually replicated in a host organism asan episome or as an integral part of the host chromosomal DNA. Ingeneral, an expression vector contains a selective marker (e.g.,ampicillin resistance, hygromycin resistance, tetracycline resistance,kanamycin resistance or neomycin resistance) to facilitate detection ofthose exogenous cells transformed with a desired DNA sequence.

Escherichia coli is an example of prokaryotic host cells that can beused to clone a polynucleotide encoding the subject antibody. Othermicrobial hosts suitable for use include Bacillus, for example, Bacillussubtilis and other species of enterobacteriaceae (such as Salmonellaspp. and Serratia spp.), and various Pseudomonas spp. In theseprokaryotic hosts, expression vectors can also be generated which willtypically contain an expression control sequence (e.g., origin ofreplication) that is compatible with the host cell. In addition, therewill be many well-known promoters, such as the lactose promoter system,the tryptophan (trp) promoter system, the beta-lactamase promoter systemor the promoter system from phage lambda. Optionally in the case ofmanipulation of a gene sequence, a promoter will usually controlexpression, and has a ribosome binding site sequence and the like toinitiate and complete transcription and translation.

Other microorganisms, such as yeast, can also be used for expression.Saccharomyces (e.g., S. cerevisiae) and Pichia are examples of suitableyeast host cells, in which a suitable vector has an expression controlsequence (e.g., promoter), an origin of replication, a terminationsequence and the like, as required. A typical promoter comprises3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeastpromoters specifically include promoters derived from alcoholdehydrogenase, isocytochrome C, and enzymes responsible for maltose andgalactose utilization.

In addition to microorganisms, mammalian cells (e.g., mammalian cellscultured in cell culture in vitro) may also be used to express theplasminogen of the present invention. See Winnacker, From Genes toClones, VCH Publishers, N.Y., N.Y. (1987). Suitable mammalian host cellsinclude CHO cell lines, various Cos cell lines, HeLa cells, myeloma celllines and transformed B cells or hybridomas. Expression vectors forthese cells may comprise an expression control sequence, such as anorigin of replication, promoter and enhancer (Queen et al. Immunol. Rev.89:49 (1986)), as well as necessary processing information sites, suchas a ribosome binding site, RNA splice site, polyadenylation site andtranscription terminator sequence. Examples of suitable expressioncontrol sequences are promoters derived from white immunoglobulin gene,SV40, adenovirus, bovine papilloma virus, cytomegalovirus and the like.See Co et al. J. Immunol. 148:1149 (1992).

Once synthesized (chemically or recombinantly), the plasminogen of thepresent invention can be purified according to standard procedures inthe art, including ammonium sulfate precipitation, affinity column,column chromatography, high performance liquid chromatography (HPLC),gel electrophoresis and the like. The plasminogen is substantially pure,e.g., at least about 80% to 85% pure, at least about 85% to 90% pure, atleast about 90% to 95% pure, or 98% to 99% pure or purer, for examplefree of contaminants such as cell debris, macromolecules other than thesubject antibody and the like.

3. Pharmaceutical Formulations

A therapeutic formulation can be prepared by mixing plasminogen of adesired purity with an optional pharmaceutical carrier, excipient orstabilizer (Remington's Pharmaceutical Sciences, 16th edition, Osol, A.ed. (1980)) to form a lyophilized preparation or an aqueous solution.Acceptable carriers, excipients and stabilizers are non-toxic to therecipient at the dosages and concentrations employed, and includebuffers, such as phosphates, citrates and other organic acids;antioxidants, including ascorbic acid and methionine; preservatives(e.g., octadecyl dimethyl benzyl ammonium chloride; hexane chloridediamine; benzalkonium chloride and benzethonium chloride; phenol,butanol or benzyl alcohol; alkyl p-hydroxybenzoates, such as methyl orpropyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol;3-pentanol; and m-cresol); low molecular weight polypeptides (less thanabout 10 residues); proteins, such as serum albumin, gelatin orimmunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone;amino acids, such as glycine, glutamine, asparagine, histidine, arginineor lysine; monosaccharides, disaccharides and other carbohydrates,including glucose, mannose or dextrins; chelating agents, such as EDTA;sugars, such as sucrose, mannitol, fucose or sorbitol; salt-formingcounterions, such as sodium; metal complexes (e.g., zinc-proteincomplexes); and/or non-ionic surfactants, such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG). Preferred lyophilized anti-VEGF antibodyformulations are described in WO 97/04801, which is incorporated hereinby reference.

The formulations of the invention may also comprise one or more activecompounds required for the particular condition to be treated,preferably those that are complementary in activity and have no sideeffects with one another, for example anti-hypertensive drugs,anti-arrhythmic drugs, drugs for treating diabetes mellitus, and thelike.

The plasminogen of the present invention may be encapsulated inmicrocapsules prepared by techniques such as coacervation or interfacialpolymerization, for example, it may be incorporated in a colloid drugdelivery system (e.g., liposomes, albumin microspheres, microemulsions,nanoparticles and nanocapsules), or incorporated inhydroxymethylcellulose or gel-microcapsules and poly-(methylmethacrylate) microcapsules in macroemulsions. These techniques aredisclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A.Ed. (1980).

The plasminogen of the present invention for in vivo administration mustbe sterile. This can be easily achieved by filtration through a sterilefiltration membrane before or after freeze drying and reconstitution.

The plasminogen of the present invention can be prepared into asustained-release preparation. Suitable examples of sustained-releasepreparations include solid hydrophobic polymer semi-permeable matriceshaving a shape and containing glycoproteins, such as films ormicrocapsules. Examples of sustained-release matrices includepolyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate)) (Langeret al. J. Biomed. Mater. Res., 15: 167-277 (1981); and Langer, Chem.Tech., 12:98-105 (1982)), or poly(vinyl alcohol), polylactides (U.S.Pat. No. 3,773,919, and EP 58,481), copolymer of L-glutamic acid and γethyl-L-glutamic acid (Sidman et al. Biopolymers 22:547(1983)),nondegradable ethylene-vinyl acetate (Langer et al. supra), ordegradable lactic acid-glycolic acid copolymers such as Lupron Depot™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly D-(−)-3-hydroxybutyric acid. Polymers,such as ethylene-vinyl acetate and lactic acid-glycolic acid, are ableto persistently release molecules for 100 days or longer, while somehydrogels release proteins for a shorter period of time. A rationalstrategy for protein stabilization can be designed based on relevantmechanisms. For example, if the aggregation mechanism is discovered tobe formation of an intermolecular S—S bond through thio-disulfideinterchange, stability is achieved by modifying sulfhydryl residues,lyophilizing from acidic solutions, controlling moisture content, usingappropriate additives, and developing specific polymer matrixcompositions.

4. Administration and Dosage

The pharmaceutical composition of the present invention is administeredin different ways, for example by intravenous, intraperitoneal,subcutaneous, intracranial, intrathecal, intraarterial (e.g., viacarotid), intramuscular, intranasal, topical or intradermaladministration or spinal cord or brain delivery. An aerosol preparation,such as a nasal spray preparation, comprises purified aqueous or othersolutions of the active agent along with a preservative and isotonicagent. Such preparations are adjusted to a pH and isotonic statecompatible with the nasal mucosa.

In some cases, the plasminogen pharmaceutical composition of the presentinvention may be modified or formulated in such a manner to provide itsability to cross the blood-brain barrier.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, and alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, or fixed oils. Intravenousvehicles include liquid and nutrient supplements, electrolytesupplements and the like. Preservatives and other additives may also bepresent, for example, such as antimicrobial agents, antioxidants,chelating agents and inert gases.

In some embodiments, the plasminogen of the invention is formulated withan agent that promotes the plasminogen to cross the blood-brain barrier.In some cases, the plasminogen of the present invention is fuseddirectly or via a linker to a carrier molecule, peptide or protein thatpromotes the fusion to cross the blood brain barrier. In someembodiments, the plasminogen of the present invention is fused to apolypeptide that binds to an endogenous blood-brain barrier (BBB)receptor. The polypeptide that is linked to plasminogen and binds to anendogenous BBB receptor promotes the fusion to cross the BBB. Suitablepolypeptides that bind to endogenous BBB receptors include antibodies(e.g., monoclonal antibodies) or antigen-binding fragments thereof thatspecifically bind to endogenous BBB receptors. Suitable endogenous BBBreceptors include, but are not limited to, insulin receptors. In somecases, antibodies are encapsulated in liposomes. See, for example, USPatent Publication No. 2009/0156498.

The medical staff will determine the dosage regimen based on variousclinical factors. As is well known in the medical field, the dosage ofany patient depends on a variety of factors, including the patient'ssize, body surface area, age, the specific compound to be administered,sex, frequency and route of administration, overall health and otherdrugs administered simultaneously. The dosage range of thepharmaceutical composition comprising plasminogen of the presentinvention may be, for example, such as about 0.0001 to 2000 mg/kg, orabout 0.001 to 500 mg/kg (such as 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg,0.75 mg/kg, 10 mg/kg and 50 mg/kg) of the subject's body weight daily.For example, the dosage may be 1 mg/kg body weight or 50 mg/kg bodyweight, or in the range of 1 mg/kg-50 mg/kg, or at least 1 mg/kg.Dosages above or below this exemplary range are also contemplated,especially considering the above factors. The intermediate dosages inthe above range are also included in the scope of the present invention.A subject may be administered with such dosages daily, every other day,weekly or based on any other schedule determined by empirical analysis.An exemplary dosage schedule includes 1-10 mg/kg for consecutive days.During administration of the drug of the present invention, thetherapeutic effect and safety of thrombosis and a thrombosis-relateddisease are required to be assessed real-timely and regularly.

5. Treatment Efficacy and Treatment Safety

One embodiment of the present invention relates to the judgment oftreatment efficacy and treatment safety after treating a subject withplasminogen. Common monitoring and assessment contents of therapeuticeffect for osteoporosis comprise follow-up survey (adverse reactions,standardized medication, basic measures, re-assessment of fracture riskfactors, etc.), new fracture assessment (clinical fracture, body heightreduction, and imageological examination), bone mineral density (BMD)measurement, and detection of bone turnover markers (BTM), comprehensivere-assessment based on these data, etc. Among them, BMD is currently themost widely used method for monitoring and assessing the therapeuticeffect. For example, BMD can be measured by means of dual energy X-rayabsorptiometry (DXA), quantitative computed tomography (QCT), singlephoton absorption measurement (SPA), or ultrasonometry. BMD can bedetected once a year after the start of treatment, and after the BMD hasstabilized, the interval may be appropriately extended, for example, toonce every 2 years. For BTM, among serological indicators, serumprocollagen type 1 N-terminal propeptide (PINP) is relatively frequentlyused at present as a bone formation indicator, and serum type 1procollagen C-terminal peptide (serum C-terminal telopeptide, S-CTX)serves as a bone resorption indicator. According to the researchprogress, more reasonable detection indicators are adjusted whereappropriate. Baseline values should be measured prior to the start oftreatment, and detections are carried out 3 months after the applicationof a formation-promoting drug therapy, and 3 to 6 months after theapplication of a resorption inhibitor drug therapy. BTM can providedynamic information of bones, is independent of BMD in effect andfunction, and is also a monitoring means complementary to BMD. Thecombination of the two has a higher clinical value. In general, if BMDrises or stabilizes after treatment, BTM has an expected change, and nofracture occurs during the treatment, the treatment response can beconsidered to be good. In addition, the present invention also relatesto the judgment of the safety of the therapeutic regimen during andafter treating a subject with plasminogen and its variants, including,but not limited to, statistics of the serum half-life, half-life oftreatment, median toxic dose (TD50) and median lethal dose (LD50) of thedrug in the body of the subject, or observing various adverse eventssuch as sensitization that occur during or after treatment.

6. Articles of Manufacture or Kits

One embodiment of the present invention relates to an article ofmanufacture or a kit comprising the plasminogen of the presentinvention. The article preferably includes a container, label or packageinsert. Suitable containers include bottles, vials, syringes and thelike. The container can be made of various materials, such as glass orplastic. The container contains a composition that is effective to treatthe disease or condition of the present invention and has a sterileaccess (for example, the container may be an intravenous solution bag orvial containing a plug that can be pierced by a hypodermic injectionneedle). At least one active agent in the composition is plasminogen.The label on or attached to the container indicates that the compositionis used for treating the aging or aging-related conditions according tothe present invention. The article may further comprise a secondcontainer containing a pharmaceutically acceptable buffer, such asphosphate buffered saline, Ringer's solution and glucose solution. Itmay further comprise other substances required from a commercial anduser perspective, including other buffers, diluents, filters, needlesand syringes. In addition, the article comprises a package insert withinstructions for use, including, for example, instructions to direct auser of the composition to administer to a patient the plasminogencomposition and other drugs for treating an accompanying disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the observed immunohistochemical results for glucagon ofthe pancreatic islets after administration of plasminogen to 24- to25-week-old diabetic mice for 35 days. A represents a normal controlgroup, B represents a control group administered with vehicle PBS, and Crepresents a group administered with plasminogen. The results show thatglucagon is expressed in the α-cell region at the periphery of thepancreatic islet in normal control mice. Compared with the groupadministered with plasminogen, glucagon-positive cells (indicated byarrow) in the control group administered with vehicle PBS are remarkablyincreased, and the positive cells infiltrate into the central region ofthe pancreatic islet; and glucagon-positive cells in the groupadministered with plasminogen are dispersed at the periphery of thepancreatic islet, and compared with the PBS group, the morphology of thepancreatic islet in the group administered with plasminogen is closer tothat of normal mice. This indicates that plasminogen can significantlyinhibit proliferation of pancreatic islet α cells and secretion ofglucagon, and correct the disordered distribution of pancreatic islet αcells, thus promoting repair of impaired pancreatic islet.

FIG. 2 shows the observed immunohistochemical results for glucagon ofthe pancreatic islets after administration of plasminogen to 26-week-olddiabetic mice for 35 days. A represents a normal control group, Brepresents the control group administered with vehicle PBS, C representsthe group administered with plasminogen, and D represents thequantitative analysis results. The results show that glucagon isexpressed in the α-cell region at the periphery of the pancreatic isletin normal control mice. Compared with the group administered withplasminogen, positive cells (indicated by arrow) in the control groupadministered with vehicle PBS are remarkably increased, theglucagon-positive cells infiltrate into the central region of thepancreatic islet, and the mean optical density quantitative analysisresults show a statistical difference (* indicates P<0.05); andglucagon-positive cells in the group administered with plasminogen aredispersed at the periphery of the pancreatic islet, and compared withthe PBS group, the morphology of the pancreatic islet in the groupadministered with plasminogen is closer to that of normal mice. Thisindicates that plasminogen can significantly inhibit proliferation ofpancreatic islet α cells and secretion of glucagon, and correct thedisordered distribution of pancreatic islet α cells, thus promotingrepair of impaired pancreatic islet.

FIG. 3 shows the observed immunohistochemical results for glucagon ofthe pancreatic islet after administration of plasminogen to mice withnormal PLG activity in a T1DM model for 28 days. A represents the blankcontrol group, B represents the control group administered with vehiclePBS, C represents the group administered with plasminogen, and Drepresents the quantitative analysis results. The results show that thepositive expression of glucagon in the control group administered withvehicle PBS is remarkably higher than that in the group administeredwith plasminogen, and the mean optical density quantitative analysisresults show that the statistical difference is significant (* indicatesP<0.05). This indicates that plasminogen can significantly reduce thesecretion of glucagon from pancreatic islet α cells in diabetic mice andpromote repair of impaired pancreatic islet.

FIG. 4 shows detection results of blood glucose after administration ofplasminogen to 24- to 25-week-old diabetic mice for 10 days and 31 days.The results show that the blood glucose level in mice in the groupadministered with plasminogen was remarkably lower than that in thecontrol group administered with vehicle PBS, and the statisticaldifference was significant (* indicates P<0.05, and ** indicatesP<0.01). In addition, with the prolongation of the administration time,the blood glucose level of the mice in the control group administeredwith vehicle PBS has a tendency to rise, while the blood glucose levelof the group administered with plasminogen gradually decreases. Thisindicates that plasminogen has a hypoglycemic effect.

FIG. 5 shows the effect of administration of plasminogen on theconcentration of serum fructosamine in diabetic mice. The detectionresults show that the concentration of serum fructosamine is remarkablydecreased after administration of plasminogen, and as compared with thatbefore administration, the statistical difference is extremelysignificant (** indicates P<0.01). This indicates that plasminogen cansignificantly reduce blood glucose in diabetic mice.

FIG. 6 shows detection results of serum fructosamine afteradministration of plasminogen to 26-week-old diabetic mice for 35 days.The detection results show that the concentration of serum fructosaminein the group administered with plasminogen is remarkably lower than thatin the control group administered with vehicle PBS, and the statisticaldifference is nearly significant (P=0.06). This indicates thatplasminogen can significantly reduce the blood glucose level in diabeticmice.

FIG. 7 shows detection results of plasma glycated hemoglobin afteradministration of plasminogen to 26-week-old diabetic mice for 35 days.The results show that the OD value of glycated hemoglobin in the mice inthe group administered with plasminogen is remarkably lower than that inthe control group administered with vehicle PBS, and the statisticaldifference is extremely significant (** indicates P<0.01). Thisindicates that plasminogen has an effect of reducing blood glucose indiabetic mice.

FIG. 8 shows detection results of IPGTT after administration ofplasminogen to 26-week-old diabetic mice for 10 days. The results showthat after intraperitoneal injection of glucose, the blood glucose levelof the mice in the group administered with plasminogen is lower thanthat in the control group administered with vehicle PBS, and comparedwith the control group administered with vehicle PBS, the glucosetolerance curve of the group administered with plasminogen is closer tothat of the normal mice group. This indicates that plasminogen canremarkably improve the glucose tolerance of diabetic mice.

FIG. 9 shows detection results of post-fasting blood glucose afteradministration of plasminogen to mice with normal PLG activity in a T1DMmodel for 10 days. The results show that the blood glucose level of themice in the control group administered with vehicle PBS is remarkablyhigher than that in the group administered with plasminogen, and thestatistical difference is extremely significant (*** indicates P<0.001).This indicates that plasminogen can significantly reduce the bloodglucose level in mice with normal PLG activity in the T1DM model.

FIG. 10 shows detection results of IPGTT after administration ofplasminogen to mice with normal PLG activity in a T1DM model for 28days. The results show that after injection of glucose, the bloodglucose concentration of the mice in the control group administered withvehicle PBS is remarkably higher than that in the group administeredwith plasminogen, and compared with the control group administered withvehicle PBS, the glucose tolerance curve of the group administered withplasminogen is closer to that of normal mice. This indicates thatplasminogen can increase the glucose tolerance of mice with normal PLGactivity in the T1DM model.

FIG. 11 shows detection results of blood glucose after administration ofplasminogen to mice in a T1DM model for 20 days. The results show thatthe blood glucose level of the mice in the control group administeredwith vehicle PBS is remarkably higher than that of the mice in the groupadministered with plasminogen, and the statistical difference issignificant (P=0.04). This indicates that plasminogen can promote theglucose decomposing ability of T1DM mice, thereby lowering bloodglucose.

FIG. 12 shows detection results of serum insulin after administration ofplasminogen to 26-week-old diabetic mice for 35 days. The results showthat the serum insulin level in the group administered with plasminogenis remarkably higher than that in the control group administered withvehicle PBS, and the statistical difference is significant (* indicatesP<0.05). This indicates that plasminogen can effectively promotesecretion of insulin.

FIG. 13 shows HE-stained images of the pancreas and the pancreatic isletarea ratios after administration of plasminogen to 24- to 25-week-olddiabetic mice for 31 days. A and B represent control groups administeredwith vehicle PBS, C and D represent groups administered withplasminogen, and E represents the quantitative analysis results ofpancreatic islet area. The results show that most of the pancreaticislets in the control groups administered with vehicle PBS areatrophied, the atrophied pancreatic islet cells are replaced by acini(indicated by ↓), and there is acinar hyperplasia at the edge of thepancreatic islets, causing the boundary between pancreatic islet andacini to be unclear; in the groups administered with plasminogen, mostof the pancreatic islets are larger than those in the control groups,there is no acinar hyperplasia in the pancreatic islets, only a smallnumber of acini remain in a few pancreatic islets, and the boundarybetween pancreatic islet and acini is clear. Comparing the groupsadministered with plasminogen with the control groups in terms of thearea ratio of pancreatic islet to pancreas, it is found that the arearatio in the administration groups are almost twice as large as that inthe control groups. This indicates that plasminogen can promote repairof impaired pancreatic islet in 24- to 25-week-old diabetic mice, bywhich diabetes mellitus is treated by repairing impaired pancreaticislet.

FIG. 14 shows the observed results of Sirius red-staining for pancreaticislets after administration of plasminogen to 24- to 25-week-olddiabetic mice for 31 days. A represents the control group administeredwith vehicle PBS, B represents the group administered with plasminogen,and C represents the quantitative analysis results. The results showedthat the collagen deposition (indicated by arrow) in the pancreaticislet of mice in the group administered with plasminogen was remarkablyless than that in the control group administered with vehicle PBS, andthe statistical difference was significant (* indicates P<0.05). Thisindicates that plasminogen can ameliorate pancreatic islet fibrosis indiabetic animals.

FIG. 15 shows the observed results of immunohistochemical staining forCaspase-3 of the pancreatic islets after administration of plasminogento 24- to 25-week-old diabetic mice for 31 days. A represents thecontrol group administered with vehicle PBS, and B represents the groupadministered with plasminogen. The results show that the expression ofCaspase-3 (indicated by arrow) in the group administered withplasminogen is remarkably lower than that in the control groupadministered with vehicle PBS. This indicates that plasminogen canreduce the apoptosis of pancreatic islet cells and protect thepancreatic tissue of diabetic mice.

FIG. 16 shows the results of immunohistochemical staining for insulin ofthe pancreatic islets after administration of plasminogen to 18-week-olddiabetic mice for 35 days. A represents the control group administeredwith vehicle PBS, B represents the group administered with plasminogen,and C represents the quantitative analysis results. The results showthat the expression of insulin (indicated by arrow) in the groupadministered with plasminogen is remarkably higher than that in thecontrol group administered with vehicle PBS, and the statisticaldifference is nearly significant (P=0.15). This indicates thatplasminogen can promote repair of pancreatic islet function and promoteproduction and secretion of insulin.

FIG. 17 shows the observed results of immunohistochemical staining forinsulin of the pancreatic islets after administration of plasminogen to24- to 25-week-old diabetic mice for 35 days. A represents the controlgroup administered with vehicle PBS, B represents the group administeredwith plasminogen, and C represents the quantitative analysis results.The results show that the expression of insulin (indicated by arrow) inthe group administered with plasminogen is remarkably higher than thatin the control group administered with vehicle PBS, and the statisticaldifference is significant (* indicates P<0.05). This indicates thatplasminogen can promote repair of pancreatic islet function and promoteproduction and secretion of insulin.

FIG. 18 shows the results of immunohistochemical staining for insulin ofthe pancreatic islets after administration of plasminogen to 26-week-olddiabetic mice for 35 days. A represents the control group administeredwith vehicle PBS, B represents the group administered with plasminogen,and C represents the quantitative analysis results. The results showthat the expression of insulin (indicated by arrow) in the groupadministered with plasminogen is remarkably higher than that in thecontrol group administered with vehicle PBS, and the statisticaldifference is extremely significant (** indicates P<0.01). Thisindicates that plasminogen can effectively promote repair of pancreaticislet function and promote production and secretion of insulin.

FIG. 19 shows the observed results of immunohistochemical staining forNF-κB of the pancreatic tissues after administration of plasminogen to24- to 25-week-old diabetic mice for 31 days. A represents a normalcontrol group, B represents the control group administered with vehiclePBS, C represents the group administered with plasminogen, and Drepresents the quantitative analysis results. The results show that theexpression of NF-κB (indicated by arrow) in the group administered withplasminogen is remarkably higher than that in the control groupadministered with vehicle PBS, and the statistical difference issignificant (* indicates P<0.05). This indicates that plasminogen canpromote expression of multi-directional nuclear transcription factorNF-κB, thereby promoting repair of an inflammation in the pancreaticislet of 24- to 25-week-old diabetic mice.

FIG. 20 shows the observed immunohistochemiscal results for glucagon ofthe pancreatic islets after administration of plasminogen to 18-week-olddiabetic mice for 35 days. A represents a normal control group, Brepresents the control group administered with vehicle PBS, C representsthe group administered with plasminogen, and D represents thequantitative analysis results. The results show that glucagon isexpressed in the α-cell region at the periphery of the pancreatic isletin normal control mice. Compared with the group administered withplasminogen, glucagon-positive cells (indicated by arrow) in the controlgroup administered with vehicle PBS are remarkably increased, theglucagon-positive cells infiltrate into the central region of thepancreatic islet, and the mean optical density quantitative analysisresults show that the statistical difference is extremely significant(** indicates P<0.01); and glucagon-positive cells in the groupadministered with plasminogen are dispersed at the periphery of thepancreatic islet, and compared with the PBS group, the morphology of thepancreatic islet in the group administered with plasminogen is closer tothat of normal mice. This indicates that plasminogen can significantlyinhibit proliferation of pancreatic islet α cells and secretion ofglucagon, and correct the disordered distribution of pancreatic islet αcells, thus promoting repair of impaired pancreatic islet.

FIG. 21 shows the observed immunohistochemical results for IRS-2 of thepancreatic islet after administration of plasminogen to 18-week-olddiabetic mice for 35 days. A represents a normal control group, Brepresents the control group administered with vehicle PBS, C representsthe group administered with plasminogen, and D represents thequantitative analysis results. The results show that the positiveexpression of IRS-2 (indicated by arrow) in the pancreatic islets ofmice in the control group administered with vehicle PBS is remarkablyless than that in the group administered with plasminogen, and thestatistical difference is extremely significant (** indicates P<0.01);and the expression level of IRS-2 in the group administered withplasminogen is closer to that of mice in the normal control group thanthat in the group administered with vehicle PBS. This indicates thatplasminogen can effectively increase expression of IRS-2 in pancreaticislet cells, improve insulin signal transduction, and reduce thepancreatic islet β cell injury in diabetic mice.

FIG. 22 shows the observed immunohistochemical results for IRS-2 of thepancreatic islets after administration of plasminogen to 24- to25-week-old diabetic mice for 31 days. A represents a normal controlgroup, B represents the control group administered with vehicle PBS, Crepresents the group administered with plasminogen, and D represents thequantitative analysis results. The results show that the positiveexpression of IRS-2 (indicated by arrow) in the pancreatic islets ofmice in the control group administered with vehicle PBS is remarkablyless than that in the group administered with plasminogen, and thestatistical difference is significant (* indicates P<0.05); and theexpression level of IRS-2 in the group administered with plasminogen iscloser to that of mice in the normal control group than that in thegroup administered with vehicle PBS. This indicates that plasminogen caneffectively increase expression of IRS-2 in pancreatic islet cells,improve insulin signal transduction, and reduce the pancreatic islet βcell injury in diabetic mice.

FIG. 23 shows the observed immunohistochemical results for IRS-2 of thepancreatic islet after administration of plasminogen to 26-week-olddiabetic mice for 35 days. A represents a normal control group, Brepresents the control group administered with vehicle PBS, C representsthe group administered with plasminogen, and D represents thequantitative analysis results. The results show that the positiveexpression of IRS-2 (indicated by arrow) in the pancreatic islets ofmice in the control group administered with vehicle PBS is remarkablylower than that in the group administered with plasminogen, and theexpression level of IRS-2 in the group administered with plasminogen iscloser to that of mice in the normal control group than that in thegroup administered with vehicle PBS. This indicates that plasminogen caneffectively increase expression of IRS-2 in pancreatic islet cells,improve insulin signal transduction, and reduce the pancreatic islet βcell injury in diabetic mice.

FIG. 24 shows the observed immunohistochemical results for IRS-2 of thepancreatic islet of T1DM mice with normal PLG activity afteradministration of plasminogen for 28 days. A represents a normal controlgroup, B represents a control group administered with vehicle PBS, and Crepresents a group administered with plasminogen. The results show thatthe positive expression of IRS-2 (indicated by arrow) in the pancreaticislets of mice in the control group administered with vehicle PBS isremarkably lower than that in the group administered with plasminogen,and the expression level of IRS-2 in the group administered withplasminogen is closer to that of mice in the normal control group thanthat in the group administered with vehicle PBS. This indicates thatplasminogen can effectively increase expression of IRS-2 in pancreaticislet cells, improve insulin signal transduction, and reduce thepancreatic islet β cell injury in T1DM mice with normal PLG activity.

FIG. 25 shows the observed immunohistochemical results for neutrophilsof the pancreatic islets after administration of plasminogen to26-week-old diabetic mice for 35 days. A represents a normal controlgroup, B represents a control group administered with vehicle PBS, and Crepresents a group administered with plasminogen. The results show thatpositive expression cells (indicated by arrow) in the group administeredwith plasminogen are less than those in the control group administeredwith vehicle PBS, and the result of the group administered withplasminogen is closer to that of the normal control group than that ofthe group administered with vehicle PBS. This indicates that plasminogencan reduce infiltration of neutrophils.

FIG. 26 shows the observed immunohistochemical results for neutrophilsof the pancreatic islets after administration of plasminogen to micewith impaired PLG activity in a T1DM model for 28 days. A represents ablank control group, B represents a control group administered withvehicle PBS, and C represents a group administered with plasminogen. Theresults show that positive expression cells (indicated by arrow) in thegroup administered with plasminogen are less than those in the controlgroup administered with vehicle PBS, and the result of the groupadministered with plasminogen is closer to that of the blank controlgroup than that of the group administered with vehicle PBS. Thisindicates that plasminogen can reduce infiltration of pancreatic isletneutrophils in mice with impaired PLG activity in a T1DM model.

FIG. 27 shows the observed immunohistochemical results for neutrophilsof the pancreatic islets after administration of plasminogen to micewith normal PLG activity in a T1DM model for 28 days. A represents ablank control group, B represents a control group administered withvehicle PBS, and C represents a group administered with plasminogen. Theresults show that positive expression cells (indicated by arrow) in thegroup administered with plasminogen are less than those in the controlgroup administered with vehicle PBS, and the result of the groupadministered with plasminogen is closer to that of the blank controlgroup than that of the group administered with vehicle PBS. Thisindicates that plasminogen can promote infiltration of pancreatic isletneutrophils in mice with normal PLG activity in a T1DM model.

FIG. 28 shows the observed immunohistochemical results for insulin ofthe pancreatic islets after administration of plasminogen to mice withimpaired PLG activity in a T1DM model for 28 days. A represents a blankcontrol group, B represents a control group administered with vehiclePBS, and C represents a group administered with plasminogen. Theimmunohistochemical results show that the positive expression of insulin(indicated by arrow) in the group administered with plasminogen isremarkably higher than that in the control group administered withvehicle PBS, and the result of the group administered with plasminogenis closer to that of the blank control group than that of the groupadministered with vehicle PBS. This indicates that plasminogen canpromote synthesis and secretion of insulin in mice with impaired PLGactivity in a T1DM model.

FIG. 29 shows the observed immunohistochemical results for insulin ofthe pancreatic islets after administration of plasminogen to mice withnormal PLG activity in a T1DM model for 28 days. A represents a blankcontrol group, B represents a control group administered with vehiclePBS, and C represents a group administered with plasminogen. Theimmunohistochemical results show that the positive expression of insulin(indicated by arrow) in the group administered with plasminogen isremarkably higher than that in the control group administered withvehicle PBS, and the result of the group administered with plasminogenis closer to that of the blank control group than that of the groupadministered with vehicle PBS. This indicates that plasminogen canpromote synthesis and expression of insulin in mice with normal PLGactivity in a T1DM model.

FIG. 30 shows the observed immunohistochemical results for NF-κB of thepancreatic islets after administration of plasminogen to mice withimpaired PLG activity in a T1DM model for 28 days. A represents a blankcontrol group, B represents a control group administered with vehiclePBS, and C represents a group administered with plasminogen. The resultsshow that the expression of NF-κB (indicated by arrow) in the groupadministered with plasminogen is remarkably higher than that in thecontrol group administered with vehicle PBS. This indicates thatplasminogen can promote expression of inflammation repair factor NF-κB,thereby promoting repair of an inflammation in the pancreatic islet.

FIG. 31 shows the observed immunohistochemical results for NF-κB of thepancreatic islet after administration of plasminogen to 18-week-olddiabetic mice for 35 days. A represents the control group administeredwith vehicle PBS, and B represents the group administered withplasminogen. The experimental results show that the expression of NF-κB(indicated by arrow) in the group administered with plasminogen isremarkably higher than that in the control group administered withvehicle PBS. This indicates that plasminogen can promote expression ofmulti-directional nuclear transcription factor NF-κB, thereby promotingrepair of an inflammation in the pancreatic islet of relatively young(18-week-old) diabetic mice.

FIG. 32 shows the observed immunohistochemical results for NF-κB of thepancreatic islet after administration of plasminogen to 26-week-olddiabetic mice for 35 days. A represents a normal control group, Brepresents a control group administered with vehicle PBS, and Crepresents a group administered with plasminogen. The results of theexperiment of the present invention show that the expression of NF-κB(indicated by arrow) in the group administered with plasminogen isremarkably higher than that in the control group administered withvehicle PBS. This indicates that plasminogen can promote expression ofmulti-directional nuclear transcription factor NF-κB, thereby promotingrepair of an inflammation in the pancreatic islet of relatively old(26-week-old) diabetic mice.

FIG. 33 shows the observed immunohistochemical results for TNF-α of thepancreatic islets after administration of plasminogen to 24- to25-week-old diabetic mice for 31 days. A represents a normal controlgroup, B represents a control group administered with vehicle PBS, and Crepresents a group administered with plasminogen. The research resultsshow that the positive expression of TNF-α (indicated by arrow) in thegroup administered with plasminogen are remarkably higher than that inthe control group administered with vehicle PBS, and the result of thegroup administered with plasminogen is closer to that of the normalcontrol group than that of the group administered with vehicle PBS. Thisindicates that plasminogen can promote expression of TNF-α, therebypromoting repair of impaired pancreatic islet in 24- to 25-week-olddiabetic mice.

FIG. 34 shows the observed immunohistochemical results for TNF-α of thepancreatic islets after administration of plasminogen to 26-week-olddiabetic mice for 31 days. A represents a normal control group, Brepresents a control group administered with vehicle PBS, and Crepresents a group administered with plasminogen. The research resultsshow that the positive expression of TNF-α (indicated by arrow) in thegroup administered with plasminogen are remarkably higher than that inthe control group administered with vehicle PBS, and the result of thegroup administered with plasminogen is closer to that of the normalcontrol group than that of the group administered with vehicle PBS. Thisindicates that plasminogen can promote expression of TNF-α, therebypromoting repair of impaired pancreatic islet in 26-week-old diabeticmice.

FIG. 35 shows the observed immunohistochemical results for TNF-α of thepancreatic islets after administration of plasminogen to mice withimpaired PLG activity in a T1DM model for 28 days. A represents thecontrol group administered with vehicle PBS, and B represents the groupadministered with plasminogen. The research results show that thepositive expression of TNF-α (indicated by arrow) in the groupadministered with plasminogen is remarkably higher than that in thecontrol group administered with vehicle PBS. This indicates thatplasminogen can promote expression of TNF-α, thereby promoting repair ofimpaired pancreatic islet in mice with impaired PLG activity in a T1DMmodel.

FIG. 36 shows the observed immunohistochemical results for IgM of thepancreatic islets after administration of plasminogen to mice withimpaired PLG activity in a T1DM model for 28 days. A represents a blankcontrol group, B represents a control group administered with vehiclePBS, and C represents a group administered with plasminogen. Theresearch results of this experiment show that the positive expression ofIgM (indicated by arrow) in the group administered with plasminogen isremarkably lower than that in the control group administered withvehicle PBS, and the result of the group administered with plasminogenis closer to that of the normal control group than that of the groupadministered with vehicle PBS. This indicates that plasminogen canreduce expression of IgM, thereby reducing impaired pancreatic islet inmice with impaired PLG activity in a T1DM model.

FIG. 37 shows the results of TUNEL staining of the pancreatic isletsafter administration of plasminogen to 24- to 25-week-old diabetic micefor 31 days. A represents a normal control group, B represents a controlgroup administered with vehicle PBS, and C represents a groupadministered with plasminogen. The results of this experiment show thatthe number of positive cells (indicated by arrow) in the groupadministered with plasminogen is remarkably smaller than that in thecontrol group administered with vehicle PBS. Positive TUNEL staining isextremely low in the normal control group. The apoptosis rate of thenormal control group is about 8%, the apoptosis rate in the groupadministered with vehicle PBS is about 93%, and the apoptosis rate inthe group administered with plasminogen is about 16%. This indicatesthat the plasminogen group can significantly reduce the apoptosis ofpancreatic islet cells in diabetic mice.

FIG. 38 shows detection results of serum insulin after administration ofplasminogen to mice in a T1DM model for 20 days. The results show thatthe concentration of serum insulin in the mice in the control groupadministered with vehicle PBS is remarkably lower than that of the micein the group administered with plasminogen, and the statisticaldifference is nearly significant (P=0.08). This indicates thatplasminogen can promote secretion of insulin in T1DM mice.

EXAMPLES Example 1. Plasminogen Reduces Proliferation of PancreaticIslet α Cells in 24- to 25-Week-Old Diabetic Mice, Restores NormalDistribution of Pancreatic Islet α Cells and Reduces Secretion ofGlucagon

Eleven male db/db mice and five male db/m mice, 24-25 weeks old, wereweighed and the db/db mice were weighed and then randomly divided intotwo groups, a group of 5 mice administered with plasminogen and acontrol group of 6 mice administered with vehicle PBS, on the day theexperiment started that was recorded as day 0; in addition, the db/mmice were used as a normal control group. Starting from day 1,plasminogen or PBS was administered. The mice in the group administeredwith plasminogen were injected with human plasminogen at a dose of 2mg/0.2 mL/mouse/day via the tail vein, and the mice in the control groupadministered with vehicle PBS was injected with an equal volume of PBSvia the tail vein or without any liquid, both lasting for 31 consecutivedays. On day 32, the mice were sacrificed, and the pancreas was takenand fixed in 4% paraformaldehyde. The fixed pancreas tissues wereparaffin-embedded after dehydration with alcohol gradient andpermeabilization with xylene. The thickness of the tissue sections was 3μm. The sections were dewaxed and rehydrated and washed with water once.The tissues were circled with a PAP pen, incubated with 3% hydrogenperoxide for 15 minutes, and washed with 0.01 M PBS twice for 5 minuteseach time. The sections were blocked with 5% normal goat serum (Vectorlaboratories, Inc., USA) for 30 minutes, and after the time was up, thegoat serum liquid was discarded. Rabbit anti-mouse glucagon antibody(Abcam) was added to the sections dropwise, incubated at 4° C.overnight, and washed with 0.01 M PBS twice for 5 minutes each time. Thesections were incubated with a secondary antibody, goat anti-rabbit IgG(HRP) antibody (Abcam), for 1 hour at room temperature and washed with0.01 M PBS twice for 5 minutes each time. The sections were developedwith a DAB kit (Vector laboratories, Inc., USA). After washed with waterthree times, the sections were counterstained with hematoxylin for 30seconds and flushed with running water for 5 minutes. After dehydrationwith alcohol gradient, permeabilization with xylenehe, and sealing witha neutral gum, the sections were observed under an optical microscope at200×.

Pancreatic islet α cells synthesize and secrete glucagon, which ismainly distributed in the peripheral region of the pancreatic islet.

The results show that compared with the group administered withplasminogen (FIG. 1C), glucagon-positive cells (indicated by arrow) inthe control group administered with vehicle PBS (FIG. 1B) are remarkablyincreased, and the positive cells infiltrate into the central region ofthe pancreatic islet; and glucagon-positive cells in the groupadministered with plasminogen are dispersed at the periphery of thepancreatic islet, and compared with the group administered with vehiclePBS, the morphology of the pancreatic islet in the group administeredwith plasminogen is closer to that in the normal control group (FIG.1A). This indicates that plasminogen can significantly inhibitproliferation of pancreatic islet α cells and secretion of glucagon in24- to 25-week-old diabetic mice, and correct the disordereddistribution of pancreatic islet α cells, suggesting that plasminogenpromotes repair of impaired pancreatic islet.

Example 2. Plasminogen Inhibits Proliferation of Pancreatic Islet αCells In 26-Week-Old Diabetic Mice, Restores Normal Distribution ofPancreatic Islet α Cells and Reduces Secretion of Glucagon

Nine male db/db mice and three male db/m mice, 26 weeks old, wereweighed and the db/db mice were weighed and then randomly divided intotwo groups, a group of 4 mice administered with plasminogen and acontrol group of 5 mice administered with vehicle PBS, on the day theexperiment started that was recorded as day 0; in addition, the db/mmice were used as a normal control group. Starting from day 1,plasminogen or PBS was administered. The mice in the group administeredwith plasminogen were injected with human plasminogen at a dose of 2mg/0.2 mL/mouse/day via the tail vein, and the mice in the control groupadministered with vehicle PBS were injected with an equal volume of PBSvia the tail vein, both lasting for 35 consecutive days. On day 36, themice were sacrificed, and the pancreas was taken and fixed in 4%paraformaldehyde. The fixed pancreas tissues were paraffin-embeddedafter dehydration with alcohol gradient and permeabilization withxylene. The thickness of the tissue sections was 3 μm. The sections weredewaxed and rehydrated and washed with water once. The tissues werecircled with a PAP pen, incubated with 3% hydrogen peroxide for 15minutes, and washed with 0.01M PBS twice for 5 minutes each time. Thesections were blocked with 5% normal goat serum (Vector laboratories,Inc., USA) for 30 minutes, and after the time was up, the goat serumliquid was discarded. Rabbit anti-mouse glucagon antibody (Abcam) wasadded to the sections dropwise, incubated at 4° C. overnight, and washedwith 0.01 M PBS twice for 5 minutes each time. The sections wereincubated with a secondary antibody, goat anti-rabbit IgG (HRP) antibody(Abcam), for 1 hour at room temperature and washed with 0.01 M PBS twicefor 5 minutes each time. The sections were developed with a DAB kit(Vector laboratories, Inc., USA). After washed with water three times,the sections were counterstained with hematoxylin for 30 seconds andflushed with running water for 5 minutes. After dehydration with alcoholgradient, permeabilization with xylenehe, and sealing with a neutralgum, the sections were observed under an optical microscope at 200×.

Pancreatic islet α cells synthesize and secrete glucagon, which ismainly distributed in the peripheral region of the pancreatic islet.

The results show that compared with the group administered withplasminogen (FIG. 2C), glucagon-positive cells (indicated by arrow) inthe control group administered with vehicle PBS (FIG. 2B) are remarkablyincreased, the positive cells infiltrate into the central region of thepancreatic islet, and the mean optical density quantitative analysisresults show a statistical difference (** indicates P<0.01) (FIG. 2D);and glucagon-positive cells in the group administered with plasminogenare dispersed at the periphery of the pancreatic islet, and comparedwith the group administered with vehicle PBS, the morphology of thepancreatic islet in the group administered with plasminogen is closer tothat in the normal control group (FIG. 2A). This indicates thatplasminogen can significantly inhibit proliferation of pancreatic isletα cells and secretion of glucagon in 26-week-old diabetic mice, andcorrect the disordered distribution of pancreatic islet α cells,suggesting that plasminogen promotes repair of impaired pancreaticislet.

Example 3. Plasminogen Reduces Secretion of Glucagon in Mice with NormalPLG Activity in T1DM Model

Fifteen 9- to 10-week-old male db/db mice with normal PLG activity wererandomly divided into three groups, a blank control group, a controlgroup administered with vehicle PBS and a group administered withplasminogen, with 5 mice in each group. The mice in the groupadministered with vehicle PBS and the group administered withplasminogen were fasted for 4 hours and then intraperitoneally injectedwith 200 mg/kg STZ (Sigma, Cat #S0130), in a single dose, to induce theT1DM model^([43]), while the blank group was not treated. 12 days afterthe injection, administration was carried out and this day was set asadministration day 1. The group administered with plasminogen wasinjected with human plasmin at a dose of 1 mg/0.1 mL/mouse/day via thetail vein, and the control group administered with vehicle PBS wasinjected with an equal volume of PBS via the tail vein, both lasting for28 consecutive days. On day 29, the mice were sacrificed, and thepancreas was taken and fixed in 4% paraformaldehyde. The fixed pancreastissues were paraffin-embedded after dehydration with alcohol gradientand permeabilization with xylene. The thickness of the tissue sectionswas 3 μm. The sections were dewaxed and rehydrated and washed with wateronce. The tissues were circled with a PAP pen, incubated with 3%hydrogen peroxide for 15 minutes, and washed with 0.01M PBS twice for 5minutes each time. The sections were blocked with 5% normal goat serum(Vector laboratories, Inc., USA) for 30 minutes, and after the time wasup, the goat serum liquid was discarded. Rabbit anti-mouse glucagonantibody (Abcam) was added to the sections dropwise, incubated at 4° C.overnight, and washed with 0.01 M PBS twice for 5 minutes each time. Thesections were incubated with a secondary antibody, goat anti-rabbit IgG(HRP) antibody (Abcam), for 1 hour at room temperature and washed with0.01 M PBS twice for 5 minutes each time. The sections were developedwith a DAB kit (Vector laboratories, Inc., USA). After washed with waterthree times, the sections were counterstained with hematoxylin for 30seconds and flushed with running water for 5 minutes. After dehydrationwith alcohol gradient, permeabilization with xylenehe, and sealing witha neutral gum, the sections were observed under an optical microscope at200×.

Pancreatic islet α cells synthesize and secrete glucagon, which ismainly distributed in the peripheral region of the pancreatic islet.

The results show that the positive expression of glucagon in the controlgroup administered with vehicle PBS (FIG. 3B) is remarkably higher thanthat in the group administered with plasminogen (FIG. 3C), and the meanoptical density quantitative analysis results show that the statisticaldifference is significant (FIG. 3D); in addition, the result of thegroup administered with plasminogen is closer to that of the blankcontrol group than that of the group administered with vehicle PBS (FIG.3A). This indicates that plasminogen can significantly reduce secretionof glucagon from pancreatic islet α cells in STZ-induced diabetic mice.

Example 4. Plasminogen Lowers Blood Glucose in Diabetic Mice

Eight 24- to 25-week-old male db/db mice were randomly divided into twogroups, a group of 5 mice administered with plasminogen, and a controlgroup of 3 mice administered with vehicle PBS. The mice were weighed andgrouped on the day when the experiment began, i.e. day 0. Starting fromthe 1st day, plasminogen or PBS was administered. The group administeredwith plasminogen was injected with human plasminogen at a dose of 2mg/0.2 mL/mouse/day via the tail vein, and the control groupadministered with vehicle PBS was injected with an equal volume of PBSvia the tail vein, both lasting for 31 consecutive days. After fastingfor 16 hours on days 10 and 31, blood glucose testing was carried outusing a blood glucose test paper (Roche, Mannheim, Germany).

The results show that the blood glucose level in mice in the groupadministered with plasminogen was remarkably lower than that in thecontrol group administered with vehicle PBS, and the statisticaldifference was significant (* indicates P<0.05, and ** indicatesP<0.01). In addition, with the prolongation of the administration time,the blood glucose level of the mice in the control group administeredwith vehicle PBS has a tendency to rise, whereas the blood glucose levelof the group administered with plasminogen gradually decreases (FIG. 4).This indicates that plasminogen has an effect of reducing blood glucosein diabetic animals.

Example 5. Plasminogen Lowers Fructosamine Level in Diabetic Mice

For five 24- to 25-week-old male db/db mice, 50 μl of blood wascollected from venous plexus in the eyeballs of each mouse one daybefore administration, recorded as day 0, for detecting a concentrationof serum fructosamine; and starting from day 1, plasminogen isadministered for 31 consecutive days. On day 32, blood was taken fromthe removed eyeballs to detect the concentration of serum fructosamine.The concentration of fructosamine was measured using a fructosaminedetection kit (A037-2, Nanjing Jiancheng).

The concentration of fructosamine reflects the average level of bloodglucose within 1 to 3 weeks. The results show that the concentration ofserum fructosamine is remarkably decreased after administration ofplasminogen, and as compared with that before administration, thestatistical difference is extremely significant (FIG. 5). This indicatesthat plasminogen can effectively reduce blood glucose in diabeticanimals.

Example 6. Plasminogen Lowers Serum Fructosamine Level in 26-Week-OldDiabetic Mice

Nine 26-week-old male db/db mice were weighed and randomly divided,according to body weight, into two groups, a group of 4 miceadministered with plasminogen and a control group of 5 mice administeredwith vehicle PBS, on the day the experiment started that was recorded asday 0. The mice in the group administered with plasminogen were injectedwith human plasminogen at a dose of 2 mg/0.2 mL/mouse/day via the tailvein, and the mice in the control group administered with vehicle PBSwas injected with an equal volume of PBS via the tail vein. Plasminogenor PBS was administered to the mice from Day 1 for 35 consecutive days.On day 36, the mice were sacrificed to detect the concentration of serumfructosamine. The concentration of fructosamine was measured using afructosamine detection kit (A037-2, Nanjing Jiancheng).

The detection results show that the concentration of serum fructosaminein the group administered with plasminogen is remarkably lower than thatin the control group administered with vehicle PBS, and the statisticaldifference is nearly significant (P=0.06) (FIG. 6). This indicates thatplasminogen can reduce blood glucose glycosamine in 26-week-old diabeticmice.

Example 7. Plasminogen Lowers Glycated Hemoglobin Level in Diabetic Mice

Nine 26-week-old male db/db mice were weighed and then randomly divided,according to body weight, into two groups, a group of 4 miceadministered with plasminogen and a control group of 5 mice administeredwith vehicle PBS, on the day the experiment started. Starting from the1st day, plasminogen or PBS was administered. The group administeredwith plasminogen was injected with human plasminogen at a dose of 2mg/0.2 mL/mouse/day via the tail vein, and the control groupadministered with vehicle PBS was injected with an equal volume of PBSvia the tail vein, both lasting for 35 consecutive days. On day 35, themice were fasted for 16 hours, and on day 36, the blood was taken fromremoved eyeballs for detecting the concentration of plasma glycatedhemoglobin.

The content of glycated hemoglobin can generally reflect the control ofblood glucose in a patient within recent 8 to 12 weeks. The results showthat the concentration of glycated hemoglobin in the mice in the groupadministered with plasminogen is remarkably lower than that in thecontrol group administered with vehicle PBS, and the statisticaldifference is significant (FIG. 7). This indicates that plasminogen caneffectively reduce the blood glucose level in diabetic animals.

Example 8. Plasminogen Improves Glucose Tolerance of Diabetic Mice

Nine 26-week-old male db/db mice and three db/m mice were involved. Onthe day the experiment started, the db/db mice were weighed and thenrandomly divided, according to body weight, into two groups, a group of4 mice administered with plasminogen and a control group of 5 miceadministered with vehicle PBS, and the db/m mice were used as a normalcontrol group. Starting from the 1st day, plasminogen or PBS wasadministered. The group administered with plasminogen was injected withhuman plasminogen at a dose of 2 mg/0.2 mL/mouse/day via the tail vein,and the control group administered with vehicle PBS was injected with anequal volume of PBS via the tail vein, both lasting for 10 consecutivedays. On day 11, after the mice were fasted for 16 hours, each mouse wasintraperitoneally injected with 5% glucose solution at 5 g/kg bodyweight, and the concentration of blood glucose was detected 0, 30, 60,90, 120, and 180 minutes using a blood glucose test paper (Roche,Mannheim, Germany).

An intraperitoneal glucose tolerance test (IPGTT) can detect thetolerance of a body to glucose. It is known in the prior art that theglucose tolerance of a diabetic patient is decreased.

The experimental results show that after intraperitoneal injection ofglucose, the blood glucose level of the mice in the group administeredwith plasminogen is lower than that in the control group administeredwith vehicle PBS, and compared with the control group administered withvehicle PBS, the glucose tolerance curve of the group administered withplasminogen is closer to that of the normal mice group (FIG. 8). Thisindicates that plasminogen can remarkably improve the glucose toleranceof diabetic mice.

Example 9. Plasminogen Lowers Blood Glucose Level in Mice with NormalPLG Activity in T1DM Model

Ten 9- to 10-week-old male db/db mice with normal PLG activity wererandomly divided into two groups, a control group administered withvehicle PBS and a group administered with plasminogen, with 5 mice ineach group. The two groups of mice were fasted for 4 hours andintraperitoneally injected with 200 mg/kg streptozotocin (STZ) (SigmaS0130), in a single dose, to induce T1DM^([43]). 12 days after theinjection of STZ, administration was carried out and this day wasrecorded as administration day 1. The group administered withplasminogen was injected with human plasmin at a dose of 1 mg/0.1mL/mouse/day via the tail vein, and the control group administered withvehicle PBS was injected with an equal volume of PBS via the tail vein,both lasting for 10 consecutive days. On day 11, after the mice werefasted for 6 hours, blood glucose testing was carried out using a bloodglucose test paper (Roche, Mannheim, Germany).

The results show that the blood glucose level of the mice in the controlgroup administered with vehicle PBS is remarkably higher than that ofthe mice in the group administered with plasminogen, and the statisticaldifference is extremely significant (FIG. 9). This indicates thatplasminogen can significantly reduce the blood glucose level in micewith normal PLG activity in the T1DM model.

Example 10. Plasminogen Improves Glucose Tolerance of T1DM Model Mice

Fifteen 9- to 10-week-old male db/db mice with normal PLG activity wererandomly divided into three groups, a blank control group, a controlgroup administered with vehicle PBS and a group administered withplasminogen, with 5 mice in each group. The mice in the groupadministered with vehicle PBS and the group administered withplasminogen were fasted for 4 hours and then intraperitoneally injectedwith 200 mg/kg STZ (Sigma S0130), in a single dose, to induceT1DM^([43]), while the blank group was not treated. 12 days after theinjection of STZ, administration was carried out and this day wasrecorded as administration day 1. The group administered withplasminogen was injected with human plasmin at a dose of 1 mg/0.1mL/mouse/day via the tail vein, and the control group administered withvehicle PBS was injected with an equal volume of PBS via the tail vein,both lasting for 28 consecutive days. On day 28, after the mice werefasted for 6 hours, 5% glucose solution was intraperitoneally injectedat 5 g/kg body weight, and the concentration of blood glucose wasdetected 0, 15, 30, 60, and 90 minutes after the injection using a bloodglucose test paper (Roche, Mannheim, Germany).

An intraperitoneal glucose tolerance test (IPGTT) can detect thetolerance of a body to glucose. It is known in the prior art that theglucose tolerance of a diabetic patient is decreased.

The results show that after injection of glucose, the blood glucoseconcentration of the mice in the control group administered with vehiclePBS is remarkably higher than that in the group administered withplasminogen, and compared with the control group administered withvehicle PBS, the glucose tolerance curve of the group administered withplasminogen is closer to that of normal mice (FIG. 10). This indicatesthat plasminogen can increase the glucose tolerance of mice with normalPLG activity in the T1DM model.

Example 11. Plasminogen Enhances Glucose Decomposing Ability of T1DMModel Mice

Eight 9- to 10-week-old male C57 mice were randomly divided into twogroups, a control group administered with vehicle PBS and a groupadministered with plasminogen, with 4 mice in each group. The mice inthe group administered with vehicle PBS and the group administered withplasminogen were fasted for 4 hours and then intraperitoneally injectedwith 200 mg/kg streptozotocin (STZ) (Sigma S0130), in a single dose, toinduce T1DM^([43]). 12 days after the injection of STZ, administrationwas carried out and this day was set as administration day 1. The groupadministered with plasminogen was injected with human plasmin at a doseof 1 mg/0.1 mL/mouse/day via the tail vein, and the control groupadministered with vehicle PBS was injected with an equal volume of PBSvia the tail vein. Administration was carried out for 19 consecutivedays. On day 20, after the mice were fasted for 6 hours, 20% glucose wasintragastrically administered at 2 g/kg body weight, and after 60minutes, blood was collected from the orbital venous plexus andcentrifuged to obtain a supernatant, which was detected for bloodglucose by means of a glucose assay kit (Rongsheng, Shanghai, 361500).

The results show that the blood glucose level of the mice in the controlgroup administered with vehicle PBS is remarkably higher than that ofthe mice in the group administered with plasminogen, and the statisticaldifference is significant (P=0.04) (FIG. 11). This indicates thatplasminogen can enhance the glucose decomposing ability of T1DM mice,thereby lowering blood glucose.

Example 12. Plasminogen Promotes Insulin Secretion Function of DiabeticMice

Nine 26-week-old male db/db mice were weighed and randomly divided,according to body weight, into two groups, a group of 4 miceadministered with plasminogen and a control group of 5 mice administeredwith vehicle PBS, on the day the experiment started that was recorded asday 0. Starting from the 1st day, plasminogen or PBS was administered.The group administered with plasminogen was injected with humanplasminogen at a dose of 2 mg/0.2 mL/mouse/day via the tail vein, andthe control group administered with vehicle PBS was injected with anequal volume of PBS via the tail vein, both lasting for 35 consecutivedays. On day 35, the mice were fasted for 16 hours; and on day 36, theblood was taken from removed eyeballs, and centrifuged to obtain asupernatant, and the serum insulin level was detected using an insulindetection kit (Mercodia AB) according to operating instructions.

The detection results show that the serum insulin level in the groupadministered with plasminogen is remarkably higher than that in thecontrol group administered with vehicle PBS, and the statisticaldifference is significant (FIG. 12). This indicates that plasminogen cansignificantly increase secretion of insulin in diabetic mice.

Example 13. Protective Effect of Plasminogen on Pancreas of DiabeticMice

Seven 24- to 25-week-old male db/db mice were weighed and randomlydivided, according to body weight, into two groups, a group of 4 miceadministered with plasminogen and a control group of 3 mice administeredwith vehicle PBS, on the day the experiment started that was recorded asday 0. Starting from the 1st day, plasminogen or PBS was administered.The group administered with plasminogen was injected with humanplasminogen at a dose of 2 mg/0.2 mL/mouse/day via the tail vein, andthe control group administered with vehicle PBS was injected with anequal volume of PBS via the tail vein, both lasting for 31 consecutivedays. On day 32, the mice were sacrificed, and the pancreas was takenand fixed in 4% paraformaldehyde. The fixed pancreas tissues wereparaffin-embedded after dehydration with alcohol gradient andpermeabilization with xylene. The tissue sections were 3 μm thick. Thesections were dewaxed and rehydrated, stained with hematoxylin and eosin(HE staining), differentiated with 1% hydrochloric acid in alcohol, andreturned to blue with ammonia water. The sections were sealed afterdehydration with alcohol gradient, and observed under an opticalmicroscope at 200× and 400×.

The results show that most of the pancreatic islets in the controlgroups administered with vehicle PBS (FIGS. 13A and 13B) are atrophied,the atrophied pancreatic islet cells are replaced by acini (indicated byarrow), and there is acinar hyperplasia at the edge of the pancreaticislets, causing the boundary between pancreatic islet and acini to beunclear; in the groups administered with plasminogen (FIGS. 13C and13D), most of the pancreatic islets are larger than those in the controlgroups, there is no acinar hyperplasia in the pancreatic islets, only asmall number of acini remain in a few pancreatic islets, and theboundary between pancreatic islet and acini is clear. Comparing theadministration groups with the control groups in terms of the area ratioof pancreatic islet to pancreas, it is found that the area ratio in theadministration groups are almost twice as large as that in the controlgroups (FIG. 13E). This indicates that plasminogen can promote repair ofimpaired pancreatic islet in diabetic mice, suggesting that plasminogenmay fundamentally cure diabetes mellitus by promoting repair of impairedpancreatic islet.

Example 14. Plasminogen Reduces Collagen Deposition in the PancreaticIslet of Diabetic Mice

Sixteen 24- to 25-week-old male db/db mice were weighed and randomlydivided, according to body weight, into two groups, a group of 10 miceadministered with plasminogen and a control group of 6 mice administeredwith vehicle PBS, on the day the experiment started that was recorded asday 0. Starting from the 1st day, plasminogen or PBS was administered.The group administered with plasminogen was injected with humanplasminogen at a dose of 2 mg/0.2 mL/mouse/day via the tail vein, andthe control group administered with vehicle PBS was injected with anequal volume of PBS via the tail vein, both lasting for 31 consecutivedays. On day 32, the mice were sacrificed, and the pancreas was takenand fixed in 4% paraformaldehyde. The fixed pancreas tissues wereparaffin-embedded after dehydration with alcohol gradient andpermeabilization with xylene. The tissue sections was 3 μm thick. Thesections were dewaxed and rehydrated and washed with water once. Afterstained with 0.1% Sirius red for 60 min, the sections were flushed withrunning water. After stained with hematoxylin for 1 min, the sectionswere flushed with running water, differentiated with 1% hydrochloricacid in alcohol and returned to blue with ammonia water, flushed withrunning water, dried and sealed. The sections were observed under anoptical microscope at 200×.

Sirius red staining allows for long-lasting staining of collagen. As aspecial staining method for pathological sections, Sirius red stainingcan show the collagen tissue specifically.

The staining results show that the collagen deposition (indicated byarrow) in the pancreatic islet of the mice in the group administeredwith plasminogen (FIG. 14B) was remarkably lower than that in thecontrol group administered with vehicle PBS (FIG. 14A), and thestatistical difference was significant (FIG. 14C). This indicates thatplasminogen can reduce pancreatic islet fibrosis in diabetic animals.

Example 15. Plasminogen Reduces Pancreatic Islet Cell Apoptosis inDiabetic Mice

Six 24- to 25-week-old male db/db mice were weighed and randomlydivided, according to body weight, into two groups, a group of 4 miceadministered with plasminogen and a control group of 2 mice administeredwith vehicle PBS, on the day the experiment started that was recorded asday 0. Starting from the 1st day, plasminogen or PBS was administered.The group administered with plasminogen was injected with humanplasminogen at a dose of 2 mg/0.2 mL/mouse/day via the tail vein, andthe control group administered with vehicle PBS was injected with anequal volume of PBS via the tail vein, both lasting for 31 consecutivedays. On day 32, the mice were sacrificed, and the pancreas was takenand fixed in 4% paraformaldehyde. The fixed pancreas tissues wereparaffin-embedded after dehydration with alcohol gradient andpermeabilization with xylene. The thickness of the tissue sections was 3μm. The sections were dewaxed and rehydrated and washed with water once.The sections were incubated with 3% hydrogen peroxide for 15 minutes andwashed with water twice for 5 minutes each time. The sections wereblocked with 5% normal goat serum liquid (Vector laboratories, Inc.,USA) for 1 hour, and thereafter, the goat serum liquid was discarded,and the tissues were circled with a PAP pen. The sections were incubatedwith rabbit anti-mouse Caspase-3 (Abcam) at 4° C. overnight and washedwith PBS twice for 5 minutes each time. The sections were incubated witha secondary antibody, goat anti-rabbit IgG (HRP) antibody (Abcam), for 1hour at room temperature and washed with PBS twice for 5 minutes eachtime. The sections were developed with a DAB kit (Vector laboratories,Inc., USA). After washed with water three times, the sections werecounterstained with hematoxylin for 30 seconds and flushed with runningwater for 5 minutes. After dehydration with a gradient, permeabilizationand sealing, the sections were observed under an optical microscope at200×.

Caspase-3 is the most important terminal cleavage enzyme in the processof cell apoptosis, and the more the expression thereof, the more thecells in an apoptotic state^([44]).

The results of the experiment of the present invention show that theexpression of Caspase-3 (indicated by arrow) in the group administeredwith plasminogen (FIG. 15B) is remarkably lower than that in the controlgroup administered with vehicle PBS (FIG. 15A). This indicates thatplasminogen can reduce the apoptosis of pancreatic islet cells.

Example 16. Plasminogen Promotes Expression and Secretion of Insulin in18-Week-Old Diabetic Mice

Eight 18-week-old male db/db mice were weighed and randomly divided,according to body weight, into two groups, a group administered withplasminogen and a control group administered with vehicle PBS, with 4mice in each group, on the day the experiment started that was recordedas day 0. Starting from the 1st day, plasminogen or PBS wasadministered. The group administered with plasminogen was injected withhuman plasminogen at a dose of 2 mg/0.2 mL/mouse/day via the tail vein,and the control group administered with vehicle PBS was injected with anequal volume of PBS via the tail vein, both lasting for 31 consecutivedays. On day 36, the mice were sacrificed, and the pancreas was takenand fixed in 4% paraformaldehyde. The fixed pancreas tissues wereparaffin-embedded after dehydration with alcohol gradient andpermeabilization with xylene. The thickness of the tissue sections was 3μm. The sections were dewaxed and rehydrated and washed with water once.The sections were incubated with 3% hydrogen peroxide for 15 minutes andwashed with water twice for 5 minutes each time. The sections wereblocked with 5% normal goat serum liquid (Vector laboratories, Inc.,USA) for 1 hour, and thereafter, the goat serum liquid was discarded,and the tissues were circled with a PAP pen. The sections were incubatedwith rabbit anti-mouse insulin antibody (Abcam) at 4° C. overnight andwashed with PBS twice for 5 minutes each time. The sections wereincubated with a secondary antibody, goat anti-rabbit IgG (HRP) antibody(Abcam), for 1 hour at room temperature and washed with PBS twice for 5minutes each time. The sections were developed with a DAB kit (Vectorlaboratories, Inc., USA). After washed with water three times, thesections were counterstained with hematoxylin for 30 seconds and flushedwith running water for 5 minutes. After gradient dehydration,permeabilization and sealing, the sections were observed under amicroscope at 200×.

The results show that the expression of insulin (indicated by arrow) inthe group administered with plasminogen (FIG. 16B) is remarkably higherthan that in the control group administered with vehicle PBS (FIG. 16A),and the statistical difference is nearly significant (P=0.15) (FIG.16C). This indicates that plasminogen can promote repair of pancreaticislet function and promote expression and secretion of insulin.

Example 17. Plasminogen Promotes Expression and Secretion of Insulin in24- to 25-Week-Old Diabetic Mice

Eight 24- to 25-week-old male db/db mice were weighed and randomlydivided, according to body weight, into two groups, a group of 5 miceadministered with plasminogen and a control group of 3 mice administeredwith vehicle PBS, on the day the experiment started that was recorded asday 0. Starting from the 1st day, plasminogen or PBS was administered.The group administered with plasminogen was injected with humanplasminogen at a dose of 2 mg/0.2 mL/mouse/day via the tail vein, andthe control group administered with vehicle PBS was injected with anequal volume of PBS via the tail vein, both lasting for 31 consecutivedays. On day 32, the mice were sacrificed, and the pancreas was takenand fixed in 4% paraformaldehyde. The fixed pancreas tissues wereparaffin-embedded after dehydration with alcohol gradient andpermeabilization with xylene. The thickness of the tissue sections was 3μm. The sections were dewaxed and rehydrated and washed with water once.The sections were incubated with 3% hydrogen peroxide for 15 minutes andwashed with water twice for 5 minutes each time. The sections wereblocked with 5% normal goat serum liquid (Vector laboratories, Inc.,USA) for 1 hour, and thereafter, the goat serum liquid was discarded,and the tissues were circled with a PAP pen. The sections were incubatedwith rabbit anti-mouse insulin antibody (Abcam) at 4° C. overnight andwashed with PBS twice for 5 minutes each time. The sections wereincubated with a secondary antibody, goat anti-rabbit IgG (HRP) antibody(Abcam), for 1 hour at room temperature and washed with PBS twice for 5minutes each time. The sections were developed with a DAB kit (Vectorlaboratories, Inc., USA). After washed with water three times, thesections were counterstained with hematoxylin for 30 seconds and flushedwith running water for 5 minutes. After gradient dehydration,permeabilization and sealing, the sections were observed under amicroscope at 200×.

The results show that the expression of insulin (indicated by arrow) inthe group administered with plasminogen is remarkably higher than thatin the control group administered with vehicle PBS, and the statisticaldifference is significant (P=0.02) (FIG. 17). This indicates thatplasminogen can effectively repair the pancreatic islet function andpromote expression and secretion of insulin.

Example 18. Plasminogen Promotes Repair of Insulin Synthesis andSecretion Function of Diabetic Mice

Nine 26-week-old male db/db mice were weighed and randomly divided,according to body weight, into two groups, a group of 4 miceadministered with plasminogen and a control group of 5 mice administeredwith vehicle PBS, on the day the experiment started that was recorded asday 0. Starting from the 1st day, plasminogen or PBS was administered.The group administered with plasminogen was injected with humanplasminogen at a dose of 2 mg/0.2 mL/mouse/day via the tail vein, andthe control group administered with vehicle PBS was injected with anequal volume of PBS via the tail vein, both lasting for 35 consecutivedays. On day 35, the mice were fasted for 16 hours; and on day 36, themice were sacrificed, and the pancreas was taken and fixed in 4%paraformaldehyde. The fixed pancreas tissues were paraffin-embeddedafter dehydration with alcohol gradient and permeabilization withxylene. The thickness of the tissue sections was 3 μm. The sections weredewaxed and rehydrated and washed with water once. The sections wereincubated with 3% hydrogen peroxide for 15 minutes and washed with watertwice for 5 minutes each time. The sections were blocked with 5% normalgoat serum liquid (Vector laboratories, Inc., USA) for 1 hour, andthereafter, the goat serum liquid was discarded, and the tissues werecircled with a PAP pen. The sections were incubated with rabbitanti-mouse insulin antibody (Abcam) at 4° C. overnight and washed withPBS twice for 5 minutes each time. The sections were incubated with asecondary antibody, goat anti-rabbit IgG (HRP) antibody (Abcam), for 1hour at room temperature and washed with PBS twice for 5 minutes eachtime. The sections were developed with a DAB kit (Vector laboratories,Inc., USA). After washed with water three times, the sections werecounterstained with hematoxylin for 30 seconds and flushed with runningwater for 5 minutes. After gradient dehydration, permeabilization andsealing, the sections were observed under a microscope at 200×.

The results show that the expression of insulin (indicated by arrow) inthe group administered with plasminogen is remarkably higher than thatin the control group administered with vehicle PBS, and the statisticaldifference is extremely significant (P=0.005) (FIG. 18). This indicatesthat plasminogen can effectively repair the pancreatic islet function ofdiabetic mice and improve expression and secretion of insulin.

Example 19. Plasminogen Promotes Expression of Multi-Directional NuclearTranscription Factor NF-κB in Pancreatic Islet of 24- to 25-Week-OldDiabetic Mice

Ten 24- to 25-week-old male db/db mice were weighed and randomlydivided, according to body weight, into two groups, a group of 4 miceadministered with plasminogen and a control group of 6 mice administeredwith vehicle PBS, on the day the experiment started that was recorded asday 0; in addition, four additional db/m mice were used as a normalcontrol group and this normal control group was not treated. Startingfrom the 1st day, plasminogen or PBS was administered. The groupadministered with plasminogen was injected with human plasminogen at adose of 2 mg/0.2 mL/mouse/day via the tail vein, and the control groupadministered with vehicle PBS was injected with an equal volume of PBSvia the tail vein, both lasting for 31 consecutive days. On day 32, themice were sacrificed, and the pancreas was taken and fixed in 4%paraformaldehyde. The fixed pancreas tissues were paraffin-embeddedafter dehydration with alcohol gradient and permeabilization withxylene. The thickness of the tissue sections was 3 μm. The sections weredewaxed and rehydrated and washed with water once. The sections wereincubated with 3% hydrogen peroxide for 15 minutes and washed with watertwice for 5 minutes each time. The sections were blocked with 5% normalgoat serum liquid (Vector laboratories, Inc., USA) for 1 hour, andthereafter, the goat serum liquid was discarded, and the tissues werecircled with a PAP pen. The sections were incubated with rabbitanti-mouse NF-κB (Abcam) at 4° C. overnight and washed with PBS twicefor 5 minutes each time. The sections were incubated with a secondaryantibody, goat anti-rabbit IgG (HRP) antibody (Abcam), for 1 hour atroom temperature and washed with PBS twice for 5 minutes each time. Thesections were developed with a DAB kit (Vector laboratories, Inc., USA).After washed with water three times, the sections were counterstainedwith hematoxylin for 30 seconds and flushed with running water for 5minutes. After gradient dehydration, permeabilization and sealing, thesections were observed under a microscope at 200×.

NF-κB is a member of the transcription factor protein family and playsan important role in the process of repairing an inflammation^([45]).

The results of the experiment of the present invention show that theexpression of NF-κB (indicated by arrow) in the group administered withplasminogen is remarkably higher than that in the control groupadministered with vehicle PBS, and the statistical difference issignificant (FIG. 19). This indicates that plasminogen can promoteexpression of multi-directional nuclear transcription factor NF-κB.

Example 20. Plasminogen Reduces Proliferation of Pancreatic Islet αCells in 18-Week-Old Diabetic Mice, Restores Normal Distribution ofPancreatic Islet α Cells and Reduces Secretion of Glucagon

Eight male db/db mice and three male db/m mice, 18 weeks old, wereweighed and the db/db mice were randomly divided, according to bodyweight, into two groups, a group administered with plasminogen and acontrol group administered with vehicle PBS, with 4 mice in each group,on the day the experiment started that was recorded as day 0; inaddition, the db/m mice were used as a normal control group. Startingfrom day 1, plasminogen or PBS was administered. The mice in the groupadministered with plasminogen were injected with human plasminogen at adose of 2 mg/0.2 mL/mouse/day via the tail vein, and the mice in thecontrol group administered with vehicle PBS were injected with an equalvolume of PBS via the tail vein, both lasting for 35 consecutive days.On day 36, the mice were sacrificed, and the pancreas was taken andfixed in 4% paraformaldehyde. The fixed pancreas tissues wereparaffin-embedded after dehydration with alcohol gradient andpermeabilization with xylene. The thickness of the tissue sections was 3μm. The sections were dewaxed and rehydrated and washed with water once.The tissues were circled with a PAP pen, incubated with 3% hydrogenperoxide for 15 minutes, and washed with 0.01M PBS twice for 5 minuteseach time. The sections were blocked with 5% normal goat serum (Vectorlaboratories, Inc., USA) for 30 minutes, and after the time was up, thegoat serum liquid was discarded. Rabbit anti-mouse glucagon antibody(Abcam) was added to the sections dropwise, incubated at 4° C.overnight, and washed with 0.01 M PBS twice for 5 minutes each time. Thesections were incubated with a secondary antibody, goat anti-rabbit IgG(HRP) antibody (Abcam), for 1 hour at room temperature and washed with0.01 M PBS twice for 5 minutes each time. The sections were developedwith a DAB kit (Vector laboratories, Inc., USA). After washed with waterthree times, the sections were counterstained with hematoxylin for 30seconds and flushed with running water for 5 minutes. After dehydrationwith alcohol gradient, permeabilization with xylenehe, and sealing witha neutral gum, the sections were observed under an optical microscope at200×.

Pancreatic islet α cells synthesize and secrete glucagon, which ismainly distributed in the peripheral region of the pancreatic islet.

The results show that compared with the group administered withplasminogen (FIG. 20C), glucagon-positive cells (indicated by arrow) inthe control group administered with vehicle PBS (FIG. 20B) areremarkably increased, the positive cells infiltrate into the centralregion of the pancreatic islet, and the mean optical densityquantitative analysis results show a statistical difference (**indicates P<0.01) (FIG. 20D); and glucagon-positive cells in the groupadministered with plasminogen are dispersed at the periphery of thepancreatic islet, and compared with the group administered with vehiclePBS, the morphology of the pancreatic islet in the group administeredwith plasminogen is closer to that in the normal control group (FIG.20A). This indicates that plasminogen can significantly inhibitproliferation of pancreatic islet α cells and secretion of glucagon in18-week-old diabetic mice, and correct the disordered distribution ofpancreatic islet α cells, suggesting that plasminogen promotes repair ofimpaired pancreatic islet.

Example 21. Plasminogen Promotes Expression of Insulin ReceptorSubstrate 2 (IRS-2) in Pancreatic Islet of 18-Week-Old Diabetic Mice

Seven male db/db mice and three male db/m mice, 18 weeks old, wereweighed and the db/db mice were randomly divided, according to bodyweight, into two groups, a group of 3 mice administered with plasminogenand a control group of 4 mice administered with vehicle PBS, on the daythe experiment started that was recorded as day 0; in addition, the db/mmice were used as a normal control group. Starting from day 1,plasminogen or PBS was administered. The mice in the group administeredwith plasminogen were injected with human plasminogen at a dose of 2mg/0.2 mL/mouse/day via the tail vein, and the mice in the control groupadministered with vehicle PBS were injected with an equal volume of PBSvia the tail vein, both lasting for 35 consecutive days. On day 36, themice were sacrificed, and the pancreas was taken and fixed in 4%paraformaldehyde. The fixed pancreas tissues were paraffin-embeddedafter dehydration with alcohol gradient and permeabilization withxylene. The thickness of the tissue sections was 3 μm. The sections weredewaxed and rehydrated and washed with water once. The tissues werecircled with a PAP pen, incubated with 3% hydrogen peroxide for 15minutes, and washed with 0.01M PBS twice for 5 minutes each time. Thesections were blocked with 5% normal goat serum (Vector laboratories,Inc., USA) for 30 minutes, and after the time was up, the goat serumliquid was discarded. Rabbit anti-mouse IRS-2 antibody (Abcam) was addedto the sections dropwise, incubated at 4° C. overnight, and washed with0.01 M PBS twice for 5 minutes each time. The sections were incubatedwith a secondary antibody, goat anti-rabbit IgG (HRP) antibody (Abcam),for 1 hour at room temperature and washed with 0.01 M PBS twice for 5minutes each time. The sections were developed with a DAB kit (Vectorlaboratories, Inc., USA). After washed with water three times, thesections were counterstained with hematoxylin for 30 seconds and flushedwith running water for 5 minutes. After dehydration with alcoholgradient, permeabilization with xylenehe, and sealing with a neutralgum, the sections were observed under an optical microscope at 200×.

Insulin receptor substrate-2 (IRS-2) is a substrate on which anactivated insulin receptor tyrosine kinase can act, is an importantmolecule in the insulin signal transduction pathway, and is veryimportant for the survival of pancreatic islet β cells. IRS-2 has aprotective effect on pancreatic islet β cells when the expressionthereof increases and is crucial for the maintenance of functionalpancreatic islet β cells^([46,47]).

The immunohistochemical results of IRS-2 show that the positiveexpression of IRS-2 (indicated by arrow) in the pancreatic islets ofmice in the control group administered with vehicle PBS (FIG. 21B) isremarkably lower than that in the group administered with plasminogen(FIG. 21C), and the statistical difference is extremely significant(FIG. 21D); in addition, the result of the group administered withplasminogen is closer to that of the blank control group than that ofthe group administered with vehicle PBS (FIG. 21A). This indicates thatplasminogen can effectively increase expression of IRS-2 in pancreaticislet cells in 18-week-old diabetic mice.

Example 22. Plasminogen Promotes Expression of IRS-2 in Pancreatic Isletof 24- to 25-Week-Old Diabetic Mice

Eleven male db/db mice and five male db/m mice, 24-25 weeks old, wereweighed and the db/db mice were randomly divided, according to bodyweight, into two groups, a group of 5 mice administered with plasminogenand a control group of 6 mice administered with vehicle PBS, on the daythe experiment started that was recorded as day 0; in addition, the db/mmice were used as a normal control group. Starting from day 1,plasminogen or PBS was administered. The mice in the group administeredwith plasminogen were injected with human plasminogen at a dose of 2mg/0.2 mL/mouse/day via the tail vein, and the mice in the control groupadministered with vehicle PBS was injected with an equal volume of PBSvia the tail vein or without any liquid, both lasting for 31 consecutivedays. On day 32, the mice were sacrificed, and the pancreas was takenand fixed in 4% paraformaldehyde. The fixed pancreas tissues wereparaffin-embedded after dehydration with alcohol gradient andpermeabilization with xylene. The thickness of the tissue sections was 3μm. The sections were dewaxed and rehydrated and washed with water once.The tissues were circled with a PAP pen, incubated with 3% hydrogenperoxide for 15 minutes, and washed with 0.01M PBS twice for 5 minuteseach time. The sections were blocked with 5% normal goat serum (Vectorlaboratories, Inc., USA) for 30 minutes, and after the time was up, thegoat serum liquid was discarded. Rabbit anti-mouse IRS-2 antibody(Abcam) was added to the sections dropwise, incubated at 4° C.overnight, and washed with 0.01 M PBS twice for 5 minutes each time. Thesections were incubated with a secondary antibody, goat anti-rabbit IgG(HRP) antibody (Abcam), for 1 hour at room temperature and washed with0.01 M PBS twice for 5 minutes each time. The sections were developedwith a DAB kit (Vector laboratories, Inc., USA). After washed with waterthree times, the sections were counterstained with hematoxylin for 30seconds and flushed with running water for 5 minutes. After dehydrationwith alcohol gradient, permeabilization with xylenehe, and sealing witha neutral gum, the sections were observed under an optical microscope at200×.

The immunohistochemical results of IRS-2 show that the positiveexpression of IRS-2 (indicated by arrow) in the pancreatic islets ofmice in the control group administered with vehicle PBS (FIG. 22B) isremarkably lower than that in the group administered with plasminogen(FIG. 22C), and the statistical difference is significant (FIG. 22D); inaddition, the result of the group administered with plasminogen iscloser to that of the normal control group than that of the groupadministered with vehicle PBS (FIG. 22A). This indicates thatplasminogen can effectively increase expression of IRS-2 in pancreaticislet cells in 24- to 25-week-old diabetic mice.

Example 23. Plasminogen Promotes Expression of IRS-2 in Pancreatic Isletof 26-Week-Old Diabetic Mice

Nine male db/db mice and three male db/m mice, 26 weeks old, wereweighed and the db/db mice were randomly divided, according to bodyweight, into two groups, a group of 4 mice administered with plasminogenand a control group of 5 mice administered with vehicle PBS, on the daythe experiment started, i.e. day 0; in addition, the db/m mice were usedas a normal control group. Starting from day 1, plasminogen or PBS wasadministered. The mice in the group administered with plasminogen wereinjected with human plasminogen at a dose of 2 mg/0.2 mL/mouse/day viathe tail vein, and the mice in the control group administered withvehicle PBS were injected with an equal volume of PBS via the tail vein,both lasting for 35 consecutive days. On day 36, the mice weresacrificed, and the pancreas was taken and fixed in 4% paraformaldehyde.The fixed pancreas tissues were paraffin-embedded after dehydration withalcohol gradient and permeabilization with xylene. The thickness of thetissue sections was 3 μm. The sections were dewaxed and rehydrated andwashed with water once. The tissues were circled with a PAP pen,incubated with 3% hydrogen peroxide for 15 minutes, and washed with0.01M PBS twice for 5 minutes each time. The sections were blocked with5% normal goat serum (Vector laboratories, Inc., USA) for 30 minutes,and after the time was up, the goat serum liquid was discarded. Rabbitanti-mouse IRS-2 antibody (Abcam) was added to the sections dropwise,incubated at 4° C. overnight, and washed with 0.01 M PBS twice for 5minutes each time. The sections were incubated with a secondaryantibody, goat anti-rabbit IgG (HRP) antibody (Abcam), for 1 hour atroom temperature and washed with 0.01 M PBS twice for 5 minutes eachtime. The sections were developed with a DAB kit (Vector laboratories,Inc., USA). After washed with water three times, the sections werecounterstained with hematoxylin for 30 seconds and flushed with runningwater for 5 minutes. After dehydration with alcohol gradient,permeabilization with xylenehe, and sealing with a neutral gum, thesections were observed under an optical microscope at 200×.

The immunohistochemical results of IRS-2 show that the positiveexpression of IRS-2 (indicated by arrow) in the pancreatic islets ofmice in the control group administered with vehicle PBS (FIG. 23B) isremarkably lower than that in the group administered with plasminogen(FIG. 23C); and The expression level of IRS-2 in the group administeredwith plasminogen is closer to that of the mice in the normal controlgroup (FIG. 23A). This indicates that plasminogen can effectivelyincrease expression of IRS-2 in pancreatic islet cells in 26-week-olddiabetic mice.

Example 24. Plasminogen Promotes Expression of IRS-2 in Pancreatic Isletof T1DM Mice with Normal PLG Activity

Fifteen 9- to 10-week-old male db/db mice with normal PLG activity wererandomly divided into three groups, a blank control group, a controlgroup administered with vehicle PBS and a group administered withplasminogen, with 5 mice in each group. The mice in the groupadministered with vehicle PBS and the group administered withplasminogen were fasted for 4 hours and then intraperitoneally injectedwith 200 mg/kg STZ (Sigma, Cat #S0130), in a single dose, to induce typeI diabetes mellitus^([43]), while the blank group was not treated. 12days after the injection, administration was carried out and this daywas set as administration day 1. The group administered with plasminogenwas injected with human plasmin at a dose of 1 mg/0.1 mL/mouse/day viathe tail vein, and the control group administered with vehicle PBS wasinjected with an equal volume of PBS via the tail vein, both lasting for28 consecutive days. On day 29, the mice were sacrificed, and thepancreas was taken and fixed in 4% paraformaldehyde. The fixed pancreastissues were paraffin-embedded after dehydration with alcohol gradientand permeabilization with xylene. The thickness of the tissue sectionswas 3 μm. The sections were dewaxed and rehydrated and washed with wateronce. The tissues were circled with a PAP pen, incubated with 3%hydrogen peroxide for 15 minutes, and washed with 0.01M PBS twice for 5minutes each time. The sections were blocked with 5% normal goat serum(Vector laboratories, Inc., USA) for 30 minutes, and after the time wasup, the goat serum liquid was discarded. Rabbit anti-mouse IRS-2antibody (Abcam) was added to the sections dropwise, incubated at 4° C.overnight, and washed with 0.01 M PBS twice for 5 minutes each time. Thesections were incubated with a secondary antibody, goat anti-rabbit IgG(HRP) antibody (Abcam), for 1 hour at room temperature and washed with0.01 M PBS twice for 5 minutes each time. The sections were developedwith a DAB kit (Vector laboratories, Inc., USA). After washed with waterthree times, the sections were counterstained with hematoxylin for 30seconds and flushed with running water for 5 minutes. After dehydrationwith alcohol gradient, permeabilization with xylenehe, and sealing witha neutral gum, the sections were observed under an optical microscope at200×.

The immunohistochemical results of IRS-2 show that the positiveexpression of IRS-2 (indicated by arrow) in the pancreatic islets ofmice in the control group administered with vehicle PBS (FIG. 24B) isremarkably lower than that in the group administered with plasminogen(FIG. 24C), and the result of the group administered with plasminogen iscloser to that of the blank control group than that of the groupadministered with vehicle PBS (FIG. 24A). This indicates thatplasminogen can effectively increase expression of IRS-2 in pancreaticislet cells in 9- to 10-week-old mice with normal PLG activity.

Example 25. Plasminogen Reduces Infiltration of Pancreatic IsletNeutrophils in 24- to 26-Week-Old Diabetic Mice

Nine male db/db mice and three male db/m mice, 24-26 weeks old, wereincluded, wherein the db/db mice were randomly divided into two groups,a group of 4 mice administered with plasminogen and a control group of 5mice administered with vehicle PBS, and the db/m mice were used as anormal control group. The day when the experiment began was recorded onDay 0, and the mice were weighed and grouped. From the second day of theexperiment, plasminogen or PBS was administered to the mice, and the daywas recorded as Day 1. The mice in the group administered withplasminogen were injected with human plasminogen at a dose of 2 mg/0.2mL/mouse/day via the tail vein, and the mice in the control groupadministered with vehicle PBS were injected with an equal volume of PBSvia the tail vein, both lasting for 35 consecutive days. On day 36, themice were sacrificed, and the pancreas was taken and fixed in 4%paraformaldehyde. The fixed pancreas tissues were paraffin-embeddedafter dehydration with alcohol gradient and permeabilization withxylene. The thickness of the tissue sections was 3 μm. The sections weredewaxed and rehydrated and washed with water once. The tissues werecircled with a PAP pen, incubated with 3% hydrogen peroxide for 15minutes, and washed with 0.01M PBS twice for 5 minutes each time. Thesections were blocked with 5% normal goat serum (Vector laboratories,Inc., USA) for 30 minutes, and after the time was up, the goat serumliquid was discarded. Rabbit anti-mouse neutrophil antibody (Abcam) wasadded to the sections dropwise, incubated at 4° C. overnight, and washedwith 0.01 M PBS twice for 5 minutes each time. The sections wereincubated with a secondary antibody, goat anti-rabbit IgG (HRP) antibody(Abcam), for 1 hour at room temperature and washed with 0.01 M PBS twicefor 5 minutes each time. The sections were developed with a DAB kit(Vector laboratories, Inc., USA). After washed with water three times,the sections were counterstained with hematoxylin for 30 seconds andflushed with running water for 5 minutes. After dehydration with alcoholgradient, permeabilization with xylenehe, and sealing with a neutralgum, the sections were observed under an optical microscope at 200×.

Neutrophils are an important member of the non-specific cellular immunesystem, and when inflammation occurs, they are attracted to the site ofinflammation by chemotactic substances.

The immunohistochemical results of neutrophils show that positiveexpression cells in the group administered with plasminogen (FIG. 25C)are less than those in the control group administered with vehicle PBS(FIG. 25B), and the result of the group administered with plasminogen iscloser to that of the normal control group (FIG. 25A) than that of thegroup administered with vehicle PBS.

Example 26. Plasminogen Reduces Infiltration of Pancreatic IsletNeutrophils in Mice with Impaired PLG Activity in T1DM Model

Ten 9- to 10-week-old male mice with impaired PLG activity were randomlydivided into three groups, a blank control group of 3 mice, a controlgroup of 3 mice administered with PBS and a group of 4 mice administeredwith plasminogen. The mice in the group administered with vehicle PBSand the group administered with plasminogen were fasted for 4 hours andthen intraperitoneally injected with 200 mg/kg STZ (Sigma S0130), in asingle dose, to induce type I diabetes mellitus^([43]), while the blankgroup was not treated. 12 days after the injection, administration wascarried out and this day was set as administration day 1. The groupadministered with plasminogen was injected with human plasmin at a doseof 1 mg/0.1 mL/mouse/day via the tail vein, and the control groupadministered with vehicle PBS was injected with an equal volume of PBSvia the tail vein, both lasting for 28 consecutive days. On day 29, themice were sacrificed, and the pancreas was taken and fixed in 4%paraformaldehyde. The fixed pancreas tissues were paraffin-embeddedafter dehydration with alcohol gradient and permeabilization withxylene. The thickness of the tissue sections was 3 μm. The sections weredewaxed and rehydrated and washed with water once. The tissues werecircled with a PAP pen, incubated with 3% hydrogen peroxide for 15minutes, and washed with 0.01M PBS twice for 5 minutes each time. Thesections were blocked with 5% normal goat serum (Vector laboratories,Inc., USA) for 30 minutes, and after the time was up, the goat serumliquid was discarded. Rabbit anti-mouse neutrophil antibody (Abcam) wasadded to the sections dropwise, incubated at 4° C. overnight, and washedwith 0.01 M PBS twice for 5 minutes each time. The sections wereincubated with a secondary antibody, goat anti-rabbit IgG (HRP) antibody(Abcam), for 1 hour at room temperature and washed with 0.01 M PBS twicefor 5 minutes each time. The sections were developed with a DAB kit(Vector laboratories, Inc., USA). After washed with water three times,the sections were counterstained with hematoxylin for 30 seconds andflushed with running water for 5 minutes. After dehydration with alcoholgradient, permeabilization with xylenehe, and sealing with a neutralgum, the sections were observed under an optical microscope at 400×.

The immunohistochemical results of neutrophils show that the positiveexpression cells (indicated by arrow) in the group administered withplasminogen (FIG. 26C) are less than those in the control groupadministered with vehicle PBS (FIG. 26B), and the result of the groupadministered with plasminogen is closer to that of the blank controlgroup (FIG. 26A) than that of the group administered with vehicle PBS.

Example 27. Plasminogen Reduces Infiltration of Pancreatic IsletNeutrophils in Mice with Normal PLG Activity in T1DM Model

Eleven 9- to 10-week-old male mice with normal PLG activity, wererandomly divided into three groups, a blank control group of 3 mice, acontrol group of 4 mice administered with vehicle PBS and a group of 4mice administered with plasminogen. The mice in the group administeredwith vehicle PBS and the group administered with plasminogen were fastedfor 4 hours and then intraperitoneally injected with 200 mg/kg STZ(Sigma S0130), in a single dose, to induce type I diabetesmellitus^([43]), while the blank group was not treated. 12 days afterthe injection, administration was carried out and this day was set asadministration day 1. The group administered with plasminogen wasinjected with human plasmin at a dose of 1 mg/0.1 mL/mouse/day via thetail vein, and the control group administered with vehicle PBS wasinjected with an equal volume of PBS via the tail vein, both lasting for28 consecutive days. On day 29, the mice were sacrificed, and thepancreas was taken and fixed in 4% paraformaldehyde. The fixed pancreastissues were paraffin-embedded after dehydration with alcohol gradientand permeabilization with xylene. The thickness of the tissue sectionswas 3 μm. The sections were dewaxed and rehydrated and washed with wateronce. The tissues were circled with a PAP pen, incubated with 3%hydrogen peroxide for 15 minutes, and washed with 0.01M PBS twice for 5minutes each time. The sections were blocked with 5% normal goat serum(Vector laboratories, Inc., USA) for 30 minutes, and after the time wasup, the goat serum liquid was discarded. Rabbit anti-mouse neutrophilantibody (Abcam) was added to the sections dropwise, incubated at 4° C.overnight, and washed with 0.01 M PBS twice for 5 minutes each time. Thesections were incubated with a secondary antibody, goat anti-rabbit IgG(HRP) antibody (Abcam), for 1 hour at room temperature and washed with0.01 M PBS twice for 5 minutes each time. The sections were developedwith a DAB kit (Vector laboratories, Inc., USA). After washed with waterthree times, the sections were counterstained with hematoxylin for 30seconds and flushed with running water for 5 minutes. After dehydrationwith alcohol gradient, permeabilization with xylenehe, and sealing witha neutral gum, the sections were observed under an optical microscope at400×.

The immunohistochemical results of neutrophils show that the positiveexpression cells (indicated by arrow) in the group administered withplasminogen (FIG. 27C) are less than those in the control groupadministered with vehicle PBS (FIG. 27B), and the result of the groupadministered with plasminogen is closer to that of the blank controlgroup (FIG. 27A) than that of the group administered with vehicle PBS.

Example 28. Plasminogen Promotes Synthesis and Secretion of Insulin inMice with Impaired PLG Activity in T1DM Model

Ten 9- to 10-week-old male mice with impaired PLG activity, wererandomly divided into three groups, a blank control group of 3 mice, acontrol group of 3 mice administered with PBS and a group of 4 miceadministered with plasminogen. The mice in the group administered withvehicle PBS and the group administered with plasminogen were fasted for4 hours and then intraperitoneally injected with 200 mg/kg STZ (SigmaS0130), in a single dose, to induce type I diabetes mellitus^([43]),while the blank group was not treated. 12 days after the injection,administration was carried out and this day was set as administrationday 1. The group administered with plasminogen was injected with humanplasmin at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and thecontrol group administered with vehicle PBS was injected with an equalvolume of PBS via the tail vein, both lasting for 28 consecutive days.On day 29, the mice were sacrificed, and the pancreas was taken andfixed in 4% paraformaldehyde. The fixed pancreas tissues wereparaffin-embedded after dehydration with alcohol gradient andpermeabilization with xylene. The thickness of the tissue sections was 3μm. The sections were dewaxed and rehydrated and washed with water once.The tissues were circled with a PAP pen, incubated with 3% hydrogenperoxide for 15 minutes, and washed with 0.01M PBS twice for 5 minuteseach time. The sections were blocked with 5% normal goat serum (Vectorlaboratories, Inc., USA) for 30 minutes, and after the time was up, thegoat serum liquid was discarded. Rabbit anti-mouse insulin antibody(Abcam) was added to the sections dropwise, incubated at 4° C.overnight, and washed with 0.01 M PBS twice for 5 minutes each time. Thesections were incubated with a secondary antibody, goat anti-rabbit IgG(HRP) antibody (Abcam), for 1 hour at room temperature and washed with0.01 M PBS twice for 5 minutes each time. The sections were developedwith a DAB kit (Vector laboratories, Inc., USA). After washed with waterthree times, the sections were counterstained with hematoxylin for 30seconds and flushed with running water for 5 minutes. After dehydrationwith alcohol gradient, permeabilization with xylenehe, and sealing witha neutral gum, the sections were observed under an optical microscope at200×.

The immunohistochemical results show that the positive expression ofinsulin (indicated by arrow) in the group administered with plasminogen(FIG. 28C) is remarkably higher than that in the control groupadministered with vehicle PBS (FIG. 28B), and the result of the groupadministered with plasminogen is closer to that of the blank controlgroup (FIG. 28A) than that of the group administered with vehicle PBS.This indicates that plasminogen can promote synthesis and secretion ofinsulin in mice with impaired PLG activity in a T1DM model.

Example 29. Plasminogen Promotes Synthesis and Expression of Insulin inMice with Normal PLG Activity in T1DM Model

Eleven 9- to 10-week-old male mice with normal PLG activity, wererandomly divided into three groups, a blank control group of 3 mice, acontrol group of 4 mice administered with vehicle PBS and a group of 4mice administered with plasminogen. The mice in the group administeredwith vehicle PBS and the group administered with plasminogen were fastedfor 4 hours and then intraperitoneally injected with 200 mg/kg STZ(Sigma S0130), in a single dose, to induce type I diabetesmellitus^([43]), while the blank group was not treated. 12 days afterthe injection, administration was carried out and this day was set asadministration day 1. The group administered with plasminogen wasinjected with human plasmin at a dose of 1 mg/0.1 mL/mouse/day via thetail vein, and the control group administered with vehicle PBS wasinjected with an equal volume of PBS via the tail vein, both lasting for28 consecutive days. On day 29, the mice were sacrificed, and thepancreas was taken and fixed in 4% paraformaldehyde. The fixed pancreastissues were paraffin-embedded after dehydration with alcohol gradientand permeabilization with xylene. The thickness of the tissue sectionswas 3 μm. The sections were dewaxed and rehydrated and washed with wateronce. The tissues were circled with a PAP pen, incubated with 3%hydrogen peroxide for 15 minutes, and washed with 0.01M PBS twice for 5minutes each time. The sections were blocked with 5% normal goat serum(Vector laboratories, Inc., USA) for 30 minutes, and after the time wasup, the goat serum liquid was discarded. Rabbit anti-mouse insulinantibody (Abcam) was added to the sections dropwise, incubated at 4° C.overnight, and washed with 0.01 M PBS twice for 5 minutes each time. Thesections were incubated with a secondary antibody, goat anti-rabbit IgG(HRP) antibody (Abcam), for 1 hour at room temperature and washed with0.01 M PBS twice for 5 minutes each time. The sections were developedwith a DAB kit (Vector laboratories, Inc., USA). After washed with waterthree times, the sections were counterstained with hematoxylin for 30seconds and flushed with running water for 5 minutes. After dehydrationwith alcohol gradient, permeabilization with xylenehe, and sealing witha neutral gum, the sections were observed under an optical microscope at200×.

The immunohistochemical results show that the positive expression ofinsulin (indicated by arrow) in the group administered with plasminogen(FIG. 29C) is remarkably higher than that in the control groupadministered with vehicle PBS (FIG. 29B), and the result of the groupadministered with plasminogen is closer to that of the blank controlgroup (FIG. 29A) than that of the group administered with vehicle PBS.This indicates that plasminogen can promote synthesis and expression ofinsulin in mice with normal PLG activity in a T1DM model.

Example 30. Plasminogen Promotes Expression of Multi-Directional NuclearTranscription Factor NF-κB in Pancreatic Islet of Mice with Impaired PLGActivity in T1DM Model

Ten 9- to 10-week-old male mice with impaired PLG activity, wererandomly divided into three groups, a blank control group of 3 mice, acontrol group of 3 mice administered with PBS and a group of 4 miceadministered with plasminogen. The mice in the group administered withvehicle PBS and the group administered with plasminogen were fasted for4 hours and then intraperitoneally injected with 200 mg/kg STZ (SigmaS0130), in a single dose, to induce type I diabetes mellitus^([43]),while the blank group was not treated. 12 days after the injection,administration was carried out and this day was set as administrationday 1. The group administered with plasminogen was injected with humanplasmin at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and thecontrol group administered with vehicle PBS was injected with an equalvolume of PBS via the tail vein, both lasting for 28 consecutive days.On day 29, the mice were sacrificed, and the pancreas was taken andfixed in 4% paraformaldehyde. The fixed pancreas tissues wereparaffin-embedded after dehydration with alcohol gradient andpermeabilization with xylene. The thickness of the tissue sections was 3μm. The sections were dewaxed and rehydrated and washed with water once.The tissues were circled with a PAP pen, incubated with 3% hydrogenperoxide for 15 minutes, and washed with 0.01M PBS twice for 5 minuteseach time. The sections were blocked with 5% normal goat serum (Vectorlaboratories, Inc., USA) for 30 minutes, and after the time was up, thegoat serum liquid was discarded. Rabbit anti-mouse NF-κB antibody (CellSignal) was added to the sections dropwise, incubated at 4° C.overnight, and washed with 0.01 M PBS twice for 5 minutes each time. Thesections were incubated with a secondary antibody, goat anti-rabbit IgG(HRP) antibody (Abcam), for 1 hour at room temperature and washed with0.01 M PBS twice for 5 minutes each time. The sections were developedwith a DAB kit (Vector laboratories, Inc., USA). After washed with waterthree times, the sections were counterstained with hematoxylin for 30seconds and flushed with running water for 5 minutes. After dehydrationwith alcohol gradient, permeabilization with xylenehe, and sealing witha neutral gum, the sections were observed under an optical microscope at200×.

As a multi-directional nuclear transcription factor, NF-κB is involvedin various gene regulations after being activated, such as cellproliferation, apoptosis, inflammation and immunity^([24]).

The experimental results show that the expression of NF-κB (indicated byarrow) in the group administered with plasminogen (FIG. 30C) isremarkably higher than that in the control group administered withvehicle PBS (FIG. 30B). This indicates that plasminogen can promoteexpression of multi-directional nuclear transcription factor NF-κB.

Example 31. Plasminogen Promotes Expression of Multi-Directional NuclearTranscription Factor NF-κB in Pancreatic Islet of 18-Week-Old DiabeticMice

Seven 18-week-old male db/db mice were weighed and randomly divided,according to body weight, into two groups, a group of 3 miceadministered with plasminogen and a control group of 4 mice administeredwith vehicle PBS, on the day the experiment started that was recorded asday 0. Starting from the 1st day, plasminogen or PBS was administered.The group administered with plasminogen was injected with humanplasminogen at a dose of 2 mg/0.2 mL/mouse/day via the tail vein, andthe control group administered with vehicle PBS was injected with anequal volume of PBS via the tail vein, both lasting for 35 consecutivedays. On day 36, the mice were sacrificed, and the pancreas was takenand fixed in 4% paraformaldehyde. The fixed pancreas tissues wereparaffin-embedded after dehydration with alcohol gradient andpermeabilization with xylene. The thickness of the tissue sections was 3μm. The sections were dewaxed and rehydrated and washed with water once.The tissues were circled with a PAP pen, incubated with 3% hydrogenperoxide for 15 minutes, and washed with 0.01M PBS twice for 5 minuteseach time. The sections were blocked with 5% normal goat serum (Vectorlaboratories, Inc., USA) for 30 minutes, and after the time was up, thegoat serum liquid was discarded. Rabbit anti-mouse NF-κB antibody (CellSignal) was added to the sections dropwise, incubated at 4° C.overnight, and washed with 0.01 M PBS twice for 5 minutes each time. Thesections were incubated with a secondary antibody, goat anti-rabbit IgG(HRP) antibody (Abcam), for 1 hour at room temperature and washed with0.01 M PBS twice for 5 minutes each time. The sections were developedwith a DAB kit (Vector laboratories, Inc., USA). After washed with waterthree times, the sections were counterstained with hematoxylin for 30seconds and flushed with running water for 5 minutes. After dehydrationwith alcohol gradient, permeabilization with xylenehe, and sealing witha neutral gum, the sections were observed under an optical microscope at200×.

The results of the experiment of the present invention show that theexpression of NF-κB (indicated by arrow) in the group administered withplasminogen (FIG. 31B) is remarkably higher than that in the controlgroup administered with vehicle PBS (FIG. 31A). This indicates thatplasminogen can promote expression of multi-directional nucleartranscription factor NF-κB.

Example 32. Plasminogen Inhibits Expression of Multi-Directional NuclearTranscription Factor NF-κB in 26-Week-Old Diabetic Mice

Nine male db/db mice and three male db/m mice, 26 weeks old, wereweighed and the db/db mice were randomly divided, according to bodyweight, into two groups, a group of 4 mice administered with plasminogenand a control group of 5 mice administered with vehicle PBS, on the daythe experiment started, i.e. day 0; in addition, the db/m mice were usedas a normal control group. Starting from the 1st day, plasminogen or PBSwas administered and this day was recorded as day 1. The groupadministered with plasminogen was injected with human plasminogen at adose of 2 mg/0.2 mL/mouse/day via the tail vein, and the control groupadministered with vehicle PBS was injected with an equal volume of PBSvia the tail vein, both lasting for 35 consecutive days. On day 36, themice were sacrificed, and the pancreas was taken and fixed in 4%paraformaldehyde. The fixed pancreas tissues were paraffin-embeddedafter dehydration with alcohol gradient and permeabilization withxylene. The thickness of the tissue sections was 3 μm. The sections weredewaxed and rehydrated and washed with water once. The tissues werecircled with a PAP pen, incubated with 3% hydrogen peroxide for 15minutes, and washed with 0.01M PBS twice for 5 minutes each time. Thesections were blocked with 5% normal goat serum (Vector laboratories,Inc., USA) for 30 minutes, and after the time was up, the goat serumliquid was discarded. Rabbit anti-mouse NF-κB antibody (Cell Signal) wasadded to the sections dropwise, incubated at 4° C. overnight, and washedwith 0.01 M PBS twice for 5 minutes each time. The sections wereincubated with a secondary antibody, goat anti-rabbit IgG (HRP) antibody(Abcam), for 1 hour at room temperature and washed with 0.01 M PBS twicefor 5 minutes each time. The sections were developed with a DAB kit(Vector laboratories, Inc., USA). After washed with water three times,the sections were counterstained with hematoxylin for 30 seconds andflushed with running water for 5 minutes. After dehydration with alcoholgradient, permeabilization with xylenehe, and sealing with a neutralgum, the sections were observed under an optical microscope at 200×.

The experimental results show that the expression of NF-κB (indicated byarrow) in the group administered with plasminogen (FIG. 32C) isremarkably higher than that in the control group administered withvehicle PBS (FIG. 32B), and the result of the group administered withplasminogen is closer to that of the normal control group (FIG. 32A)than that of the group administered with vehicle PBS. This indicatesthat plasminogen can promote expression of multi-directional nucleartranscription factor NF-κB in relatively old (26-week-old) diabeticmice.

Example 33. Plasminogen Promotes Expression of TNF-α in Pancreatic Isletof 24- to 25-Week-Old Diabetic Mice

Eleven male db/db mice and five male db/m mice, 24-25 weeks old, wereweighed and the db/db mice were randomly divided, according to bodyweight, into two groups, a group of 5 mice administered with plasminogenand a control group of 6 mice administered with vehicle PBS, on the daythe experiment started that was recorded as day 0; in addition, the db/mmice were used as a normal control group. Starting from the 1st day,plasminogen or PBS was administered. The group administered withplasminogen was injected with human plasminogen at a dose of 2 mg/0.2mL/mouse/day via the tail vein, and the control group administered withvehicle PBS was injected with an equal volume of PBS via the tail veinor without any liquid, both lasting for 31 consecutive days. On day 32,the mice were sacrificed, and the pancreas was taken and fixed in 4%paraformaldehyde. The fixed pancreas tissues were paraffin-embeddedafter dehydration with alcohol gradient and permeabilization withxylene. The thickness of the tissue sections was 3 μm. The sections weredewaxed and rehydrated and washed with water once. The tissues werecircled with a PAP pen, incubated with 3% hydrogen peroxide for 15minutes, and washed with 0.01M PBS twice for 5 minutes each time. Thesections were blocked with 5% normal goat serum (Vector laboratories,Inc., USA) for 30 minutes, and after the time was up, the goat serumliquid was discarded. Rabbit anti-mouse TNF-α antibody (Abcam) was addedto the sections dropwise, incubated at 4° C. overnight, and washed with0.01 M PBS twice for 5 minutes each time. The sections were incubatedwith a secondary antibody, goat anti-rabbit IgG (HRP) antibody (Abcam),for 1 hour at room temperature and washed with 0.01 M PBS twice for 5minutes each time. The sections were developed with a DAB kit (Vectorlaboratories, Inc., USA). After washed with water three times, thesections were counterstained with hematoxylin for 30 seconds and flushedwith running water for 5 minutes. After dehydration with alcoholgradient, permeabilization with xylenehe, and sealing with a neutralgum, the sections were observed under an optical microscope at 200×.

Tumor necrosis factor-α (TNF-α) is mainly produced by activatedmonocytes/macrophages and is an important pro-inflammatoryfactor^([48]).

The research results of this experiment show that the positiveexpression of TNF-α in the group administered with plasminogen (FIG.33C) are remarkably higher than that in the control group administeredwith vehicle PBS (FIG. 33B), and the result of the group administeredwith plasminogen is closer to that of the normal control group (FIG.33A) than that of the group administered with vehicle PBS. Thisindicates that plasminogen can promote expression of TNF-α in 24- to25-week-old diabetic mice.

Example 34. Plasminogen Inhibits Expression of TNF-α in Pancreatic Isletof 26-Week-Old Diabetic Mice

Nine male db/db mice and three male db/m mice, 26 weeks old, wereweighed and the db/db mice were randomly divided, according to bodyweight, into two groups, a group of 4 mice administered with plasminogenand a control group of 5 mice administered with vehicle PBS, on the daythe experiment started, i.e. day 0; in addition, the db/m mice were usedas a normal control group. Starting from day 1, plasminogen or PBS wasadministered. The mice in the group administered with plasminogen wereinjected with human plasminogen at a dose of 2 mg/0.2 mL/mouse/day viathe tail vein, and the mice in the control group administered withvehicle PBS was injected with an equal volume of PBS via the tail veinor without any liquid, both lasting for 35 consecutive days. On day 36,the mice were sacrificed, and the pancreas was taken and fixed in 4%paraformaldehyde. The fixed pancreas tissues were paraffin-embeddedafter dehydration with alcohol gradient and permeabilization withxylene. The thickness of the tissue sections was 3 μm. The sections weredewaxed and rehydrated and washed with water once. The tissues werecircled with a PAP pen, incubated with 3% hydrogen peroxide for 15minutes, and washed with 0.01M PBS twice for 5 minutes each time. Thesections were blocked with 5% normal goat serum (Vector laboratories,Inc., USA) for 30 minutes, and after the time was up, the goat serumliquid was discarded. Rabbit anti-mouse TNF-α antibody (Abcam) was addedto the sections dropwise, incubated at 4° C. overnight, and washed with0.01 M PBS twice for 5 minutes each time. The sections were incubatedwith a secondary antibody, goat anti-rabbit IgG (HRP) antibody (Abcam),for 1 hour at room temperature and washed with 0.01 M PBS twice for 5minutes each time. The sections were developed with a DAB kit (Vectorlaboratories, Inc., USA). After washed with water three times, thesections were counterstained with hematoxylin for 30 seconds and flushedwith running water for 5 minutes. After dehydration with alcoholgradient, permeabilization with xylenehe, and sealing with a neutralgum, the sections were observed under an optical microscope at 200×.

The research results show that the positive expression of TNF-α in thegroup administered with plasminogen (FIG. 34C) are remarkably higherthan that in the control group administered with vehicle PBS (FIG. 34B),and the result of the group administered with plasminogen is closer tothat of the normal control group (FIG. 34A) than that of the groupadministered with vehicle PBS. This indicates that plasminogen canpromote expression of TNF-α in 26-week-old diabetic mice.

Example 35. Plasminogen Promotes Expression of TNF-α in Pancreatic Isletof Mice with Impaired PLG Activity in T1DM Model

Seven 9- to 10-week-old male mice with impaired PLG activity wererandomly divided into two groups, a control group of 3 mice administeredwith PBS and a group of 4 mice administered with plasminogen. The twogroups of mice were fasted for 4 hours and intraperitoneally injectedwith 200 mg/kg STZ (Sigma S0130), in a single dose, to induce type Idiabetes mellitus^([43]). 12 days after the injection, administrationwas carried out and this day was set as administration day 1. The groupadministered with plasminogen was injected with human plasmin at a doseof 1 mg/0.1 mL/mouse/day via the tail vein, and the control groupadministered with vehicle PBS was injected with an equal volume of PBSvia the tail vein, both lasting for 28 consecutive days. On day 29, themice were sacrificed, and the pancreas was taken and fixed in 4%paraformaldehyde. The fixed pancreas tissues were paraffin-embeddedafter dehydration with alcohol gradient and permeabilization withxylene. The thickness of the tissue sections was 3 μm. The sections weredewaxed and rehydrated and washed with water once. The tissues werecircled with a PAP pen, incubated with 3% hydrogen peroxide for 15minutes, and washed with 0.01M PBS twice for 5 minutes each time. Thesections were blocked with 5% normal goat serum (Vector laboratories,Inc., USA) for 30 minutes, and after the time was up, the goat serumliquid was discarded. Rabbit anti-mouse antibody TNF-α (Abcam) was addedto the sections dropwise, incubated at 4° C. overnight, and washed with0.01 M PBS twice for 5 minutes each time. The sections were incubatedwith a secondary antibody, goat anti-rabbit IgG (HRP) antibody (Abcam),for 1 hour at room temperature and washed with 0.01 M PBS twice for 5minutes each time. The sections were developed with a DAB kit (Vectorlaboratories, Inc., USA). After washed with water three times, thesections were counterstained with hematoxylin for 30 seconds and flushedwith running water for 5 minutes. After dehydration with alcoholgradient, permeabilization with xylenehe, and sealing with a neutralgum, the sections were observed under an optical microscope at 200×.

The research results of this experiment show that the positiveexpression of TNF-α in the group administered with plasminogen (FIG.35B) is remarkably higher than that in the control group administeredwith vehicle PBS (FIG. 35A).

This indicates that plasminogen can promote expression of TNF-α in micewith impaired PLG activity in a T1DM model.

Example 36. Plasminogen Alleviates Impaired Pancreatic Islet in Micewith Impaired PLG Activity in T1DM Model

Ten 9- to 10-week-old male mice with impaired PLG activity, wererandomly divided into three groups, a blank control group of 3 mice, acontrol group of 3 mice administered with PBS and a group of 4 miceadministered with plasminogen. The mice in the group administered withvehicle PBS and the group administered with plasminogen were fasted for4 hours and then intraperitoneally injected with 200 mg/kg STZ (SigmaS0130), in a single dose, to induce type I diabetes mellitus^([43]),while the blank group was not treated. 12 days after the injection,administration was carried out and this day was set as administrationday 1. The group administered with plasminogen was injected with humanplasmin at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and thecontrol group administered with vehicle PBS was injected with an equalvolume of PBS via the tail vein, both lasting for 28 consecutive days.On day 29, the mice were sacrificed, and the pancreas was taken andfixed in 4% paraformaldehyde. The fixed pancreas tissues wereparaffin-embedded after dehydration with alcohol gradient andpermeabilization with xylene. The thickness of the tissue sections was 3μm. The sections were dewaxed and rehydrated and washed with water once.The tissues were circled with a PAP pen, incubated with 3% hydrogenperoxide for 15 minutes, and washed with 0.01M PBS twice for 5 minuteseach time. The sections were blocked with 5% normal goat serum (Vectorlaboratories, Inc., USA) for 30 minutes, and after the time was up, thegoat serum liquid was discarded. Goat anti-mouse IgM (HRP) antibody(Abcam) was added to the sections dropwise, incubated for 1 hour at roomtemperature and washed with 0.01 M PBS twice for 5 minutes each time.The sections were developed with a DAB kit (Vector laboratories, Inc.,USA). After washed with water three times, the sections werecounterstained with hematoxylin for 30 seconds and flushed with runningwater for 5 minutes. After dehydration with alcohol gradient,permeabilization with xylenehe, and sealing with a neutral gum, thesections were observed under an optical microscope at 200×.

IgM antibodies play an important role during the clearance of apoptoticand necrotic cells, and the local level of IgM antibodies at the injurysite in tissues and organs are positively correlated with the degree ofinjury^([49,50]) Therefore, detection of local level of IgM antibodiesin tissues and organs can reflect the injury of the tissues and organs.

The research results show that the positive expression of IgM in thegroup administered with plasminogen (FIG. 36C) is remarkably lower thanthat in the control group administered with vehicle PBS (FIG. 36B), andthe result of the group administered with plasminogen is closer to thatof the blank control group (FIG. 36A) than that of the groupadministered with vehicle PBS. This indicates that plasminogen canreduce expression of IgM, suggesting that plasminogen can alleviateimpaired pancreatic islet in mice with impaired PLG activity in a T1DMmodel.

Example 37. Plasminogen Reduces Pancreatic Islet Cell Apoptosis in 24-to 25-Week-Old Diabetic Mice

Eleven male db/db mice and five male db/m mice, 24-25 weeks old, wereweighed and the db/db mice were randomly divided, according to bodyweight, into two groups, a group of 5 mice administered with plasminogenand a control group of 6 mice administered with vehicle PBS, on the daythe experiment started that was recorded as day 0; in addition, the db/mmice were used as a normal control group. Starting from the 1st day,plasminogen or PBS was administered. The group administered withplasminogen was injected with human plasminogen at a dose of 2 mg/0.2mL/mouse/day via the tail vein, and the control group administered withvehicle PBS was injected with an equal volume of PBS via the tail veinor without any liquid, both lasting for 31 consecutive days. On day 32,the mice were sacrificed, and the pancreas was taken and fixed in 4%paraformaldehyde. The fixed pancreas tissues were paraffin-embeddedafter dehydration with alcohol gradient and permeabilization withxylene. The thickness of the tissue sections was 3 μm. The sections weredewaxed and rehydrated and washed with water once. A tissue was circledwith a PAP pen, and a proteinase K solution was added dropwise to coverthe tissue, incubated at room temperature for 7 min, and washed threetimes with 0.01 M PBS for 3 minutes each time. A mixed liquid of reagent1 and reagent 2 (5:45) of TUNEL kit (Roche) was added to the sectionsdropwise, incubated at a constant temperature of 37° C. for 40 min, andwashed with 0.01 M PBS three times for 3 minutes each time. A 3%hydrogen peroxide aqueous solution (hydrogen peroxide:methanol=1:9)prepared by using methanol was added to the sections dropwise, incubatedat room temperature for 20 minutes in the dark, and washed with 0.01 MPBS three times for 3 minutes each time. A tunel kit reagent 3 was addedto the sections dropwise, incubated at a constant temperature of 37° C.for 30 min, and washed with 0.01 M PBS three times. A DAB kit (Vectorlaboratories, Inc., USA) was applied for development. After washed withwater three times, counterstaining was carried out with hematoxylin for30 seconds followed by rinsing with running water for 5 minutes. Afterdehydration with alcohol gradient, permeabilization with xylenehe, andsealing with a neutral gum, the sections were observed under an opticalmicroscope at 200×.

TUNEL staining may be used to detect the breakage of nuclear DNA intissue cells during the late stage of apoptosis.

The results of this experiment show that the number of positive cells(indicated by arrow) in the group administered with plasminogen (FIG.37C) is remarkably smaller than that in the control group administeredwith vehicle PBS (FIG. 37B). Positive TUNEL staining is extremely low inthe normal control group (FIG. 37A). The apoptosis rate of the normalcontrol group is about 8%, the apoptosis rate in the group administeredwith vehicle PBS is about 93%, and the apoptosis rate in the groupadministered with plasminogen is about 16%. This indicates that theplasminogen group can significantly reduce the apoptosis of pancreaticislet cells in diabetic mice.

Example 38. Plasminogen Improves Secretion of Insulin in T1DM Model Mice

Six 9- to 10-week-old male C57 mice were randomly divided into twogroups, a control group administered with vehicle PBS and a groupadministered with plasminogen, with 3 mice in each group. The two groupsof mice were fasted for 4 hours and intraperitoneally injected with 200mg/kg streptozotocin (STZ) (Sigma S0130), in a single dose, to induceT1DM^([43]). 12 days after the injection of STZ, administration wascarried out and this day was set as administration day 1. The groupadministered with plasminogen was injected with human plasmin at a doseof 1 mg/0.1 mL/mouse/day via the tail vein, and the control groupadministered with vehicle PBS was injected with an equal volume of PBSvia the tail vein. Administration was carried out for 20 consecutivedays. On day 21, the mice were fasted for 6 hours, and then, blood wastaken from venous plexus in the eyeballs, the blood was centrifuged toobtain a supernatant, and the concentration of serum insulin wasdetected using an insulin detection kit (Mercodia AB) according tooperating instructions.

The results show that the concentration of insulin in the mice in thecontrol group administered with vehicle PBS is remarkably lower thanthat of the mice in the group administered with plasminogen, and thestatistical difference is nearly significant (P=0.08) (FIG. 38). Thisindicates that plasminogen can promote secretion of insulin in T1DMmice.

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The invention claimed is:
 1. A method for reducing blood glucose in adiabetic subject, comprising administering an effective amount ofplasminogen to the subject, thereby reducing blood glucose in thesubject.
 2. The method of claim 1, wherein the blood glucose is selectedfrom one or more of: a serum glucose level, a serum fructosamine level,and a serum glycated hemoglobin level.
 3. The method of claim 1, whereinthe diabetes mellitus is T1DM or T2DM.
 4. The method of claim 1, whereinthe plasminogen is administered in combination with one or more otherdrugs or therapies.
 5. The method of claim 4, wherein the plasminogen isadministered in combination with one or more drugs selected fromanti-diabetic drugs, drugs against cardiovascular and cerebrovasculardiseases, anti-thrombotic drugs, anti-hypertensive drugs, antilipemicdrugs, anticoagulant drugs, and anti-infective drugs.
 6. The method ofclaim 1, wherein the plasminogen has at least 75%, 80%, 85%, 90%, 95%,96%, 97%, 98% or 99% sequence identity with SEQ ID NO. 2, and still hasthe plasminogen activity.
 7. The method of claim 1, wherein theplasminogen is a protein that comprises a plasminogen active fragmentand still has the plasminogen activity.
 8. The method of claim 1,wherein the plasminogen is selected from Glu-plasminogen,Lys-plasminogen, mini-plasminogen, micro-plasminogen, delta-plasminogenor their variants that retain the plasminogen activity.
 9. The method ofclaim 1, wherein the plasminogen is a natural or synthetic humanplasminogen.
 10. The method of claim 1, wherein the plasminogen isadministered to the subject at a dosage of 1-100 mg/kg.
 11. The methodof claim 10, wherein the dosage of the plasminogen is repeated at leastonce.
 12. The method of claim 10, wherein the plasminogen isadministered at least daily.
 13. The method of claim 1, wherein thesubject is human.
 14. A method for treating diabetes mellitus in asubject, comprising administering an effective amount of plasminogen tothe subject to reduce secretion of glucagon in the subject after eating,wherein the plasminogen achieves a return to a normal or nearly normallevel of blood glucose in the subject by promoting expression and/orsecretion of insulin while reducing expression and/or secretion ofglucagon in the subject, thereby treating diabetes mellitus in thesubject.
 15. The method of claim 14, wherein the blood glucose ispostprandial blood glucose.
 16. A method for promoting the utilizationof glucose in a diabetic subject, comprising administering an effectiveamount of plasminogen to the subject, thereby promoting the utilizationof glucose in the subject.
 17. The method of claim 16, wherein theplasminogen improves the glucose tolerance in the diabetic subject. 18.A method for treating diabetes mellitus in a subject, comprisingadministering an effective amount of plasminogen to the subject toreduce secretion of glucagon in the subject after eating, wherein theplasminogen promotes expression and/or secretion of insulin whilereducing expression and/or secretion of glucagon in the subject, therebytreating diabetes mellitus in the subject.